Patent Application
20110160081
The present invention relates to, inter alia, a biological reagent comprising a complex of G protein-coupled receptors (GPCR), methods of producing the same, methods for determining oligomeric GPCR interactions, and methods for identifying compounds that interact with GPCR oligomers, e.g., dimers.
G protein-coupled receptors comprise a diverse, well-studied system for transducing signals from the extracellular milieu to a variety of intracellular signaling molecules (1). Although GPCRs have been recently considered to be oligomers such as dimers in the plasma membrane (2), understanding of the structural details and functional role of this spatial organization is still limited (3). Most importantly, it has not been established whether activation of Class A rhodopsin-like GPCRs is affected by such an organization in a particular quaternary structure. Recently both rhodopsin (4) and the β2-adrenergic receptor (B2AR) (5) have been shown to signal efficiently to G proteins when reconstituted into lipid nanodiscs containing only a single receptor. Thus, after solubilization and reconstitution, these GPCRs can function alone. However, such studies cannot clarify whether these receptors do function alone in vivo, and this question still needs to be addressed directly through an exploration of their native organization.
The Class C heterodimeric GABAB receptor has been shown to function as a dimer through a “transactivation” mechanism in which agonist binding to one protomer signals through the second protomer to G protein (6). A clever adaptation of the endoplasmic reticulum (ER) retention signal from the GABAB receptor has enabled controlled cell surface expression and study of signaling by defined metabotropic glutamate receptor (mGluR) “hetero”-dimers (7), which have been shown to signal through both trans- and cis-activation (7). Such an approach to engineered ER retention signals has not yet been successful in Class A receptors, but Class A glycoprotein hormone receptors with large N-terminal binding sites also appear to be capable of both trans- as well as cis-activation (8).
The native functional signaling unit in other Class A rhodopsin-like receptors remains unclear. A number of studies have shown that coexpression of two different Class A GPCRs can lead to signaling properties that differ from their properties when expressed alone (9, 10). This could result from downstream signaling crosstalk or from a heteromeric signaling unit, which would require communication between the protomers. A conformational change at the dimer interface has been associated with activation (13). In addition, agonist binding to a single protomer of the rhodopsin-like leukotriene B4 receptor BLT1 induced asymmetric conformational changes within the dimer, consistent with transfer of information between the protomers (113). In contrast to the GABAB receptor and TSH and LH receptors, the data for BLT1 support the existence of cis- but not trans-activation.
This proposed asymmetric nature of the signaling unit might account in full or in part for the negative cooperativity that has been observed for ligand binding in class A GPCRs. For example, in cells expressing chemokine receptor heterodimers, a selective ligand for protomer 1 can lead to dissociation of a ligand prebound to protomer 2 (96), consistent with transmittal of an altered conformation across the dimer interface.
Receptor-G protein fusion constructs, in which the C-terminus of a GPCR is fused to the N-terminus of a Ga protein, have been widely used to explore receptor signaling (14-20). Coexpression of such GPCR-G protein fusions with a second GPCR has been used to study heterodimer signaling; in such a scenario the unfused GPCR can activate the G protein fused to a coexpressed GPCR (16-20). However, coexpression of GPCRs is likely to lead to a combination of different signaling units consisting of both homodimers and heterodimers, which makes it difficult to study the functional interactions between two receptors in a defined heteromeric signaling unit. Indeed, it has been shown that tethered G proteins fused to a single membrane-spanning segment can be activated efficiently by a coexpressed GPCR (16, 21), suggesting that a GPCR-G protein fusion construct also might provide G protein for activation by another receptor without actually participating in the relevant dimeric signaling unit. The long GPCR cytoplasmic tails and flexible linkers to which G proteins have been fused are likely to lead to promiscuous interactions that exacerbate this problem. Indeed, the tether attaching the B2AR to fused G proteins can be dramatically shortened with preserved function (22), but whether the G protein in this case is activated by its own receptor or another receptor is not known.
The catecholamine dopamine plays a major role in the regulation of cognitive, emotional and behavioral functions, and abnormalities in its regulation have been implicated in a number of psychiatric and neurological disorders. Dopamine acts through D2-like (D2, D3, D4) and D1-like (D1, D5) receptors, which are members of the seven transmembrane segment GPCR superfamily. Many drugs used to treat psychiatric disorders, including schizophrenia, attention-deficit hyperactivity disorder (ADHD), and depression, target dopamine receptors, either directly or indirectly. That dopamine receptors may exist and function in complex with other GPCRs opens new pharmacological possibilities that will be best exploited if based on a clear understanding of the mechanistic basis of this signaling crosstalk.
What is most physiologically relevant is understanding the role of the oligomeric, e.g., dimeric organization of GPCRs in signaling (1, 3). Indeed, one of the great challenges in GPCR biology today is the weak mechanistic link between the physical interaction of receptors in the membrane and signaling crosstalk of presumed heterodimers or hetero-oligomers. There is a great deal of evidence from many laboratories that many GPCRs interact as heterodimers (61, 62). A number of findings, now support the existence of higher order homo-oligomers (63-66). This raises the possibility that GPCR heteromers may interact not as heterodimers per se but rather as higher order hetero-oligomers composed of homodimer subunits.
A large number of studies have demonstrated signaling cross-talk between coexpressed GPCRs (67). In almost all cases, however, the mechanistic link between heteromerization and signaling is tenuous. Although activation of two co-expressed receptors may be essential, signaling crosstalk could nonetheless take place downstream of parallel homomeric receptor-mediated G protein activation and in such a case would not be a direct result of heteromeric signaling. Such a downstream crosstalk mechanism, while often ignored, is very difficult to rule out. One example of this complexity is a recent study of a presumed dopamine D1-D2 receptor heterodimer that has been carried out both in heterologous cells (68) and in the brain (69). These receptors appear to be co-expressed in some neurons in vivo (69). In heterologous cells, they have been inferred to physically interact based on fluorescence resonance energy transfer (FRET) (70, 71) as well as co-internalization (72, 73) and co-retention of mutants (74). Activating both dopamine D1 receptor (D1R) and dopamine D2 receptors (D2R) leads to altered signaling and recruitment of Gq-mediated signaling (68, 69), whereas D1R signaling is normally Gs/olf mediated and D2R signaling is normally Go/i mediated. These findings are intriguing and open exciting avenues of drug design targeted at selective heteromers (75). In this study, however, D1R-mediated Gq signaling was observed in the brain (76, 77), but in other studies, it has been shown to be insensitive to D2R blockade (78), suggesting a role for other cellular factors in the coupling of D1R to the Gq pathway. That D2R signaling appears to be essential in one case and not in the other suggests a complex interaction of signaling mechanisms. Evidence for a priming effect for D1R-mediated Gq signaling is an example of such a potential mechanism (79, 80).
D2R has also been reported to interact with the dopamine D3 receptor (D3R), and coexpression of the D2 and D3 receptors has been reported to modulate the function of both receptors (81-83). More recently the D2R has been shown to modulate and to physically associate with the dopamine transporter as well (84, 85).
In addition to its reported interactions with receptors from the dopamine subfamily, there is a substantial literature on heteromerization of D2R with multiple other Family A receptors. There is evidence for direct physical interaction between D2R and the somatostatin subtype 5 receptor (SSTR5) (86), D2R and adenosine A2A receptor (87, 88), and D2R and CB1 cannabinoid receptor (89). In each of these cases, changes in signaling were observed upon receptor coexpression, with either altered D2R pharmacology by the partner protomer and/or an alteration in the properties of the partner in response to drugs acting at the D2R. In the case of the D2R-CB1 heteromer, dual-agonist mediated activation of Gs was reported, although neither receptor alone is able to activate this Ga subunit (89). These results are intriguing and suggest the possibility of an untapped level of pharmacological diversity for new compound development, as well as a host of potential roles for in vivo signaling specificity for these putative heteromers. However, in none of these studies is it possible to rule out downstream signaling crosstalk and thus to establish incontrovertibly that direct signaling by the D2R heteromer is responsible for the crosstalk.
Such a mechanistic interrogation of heteromeric signaling in Family A GPCRs has been difficult. As mentioned above, mechanistic understanding of the functional role of GPCR dimerization is more advanced in the Family C receptors, due, in part, to the availability of a clever adaptation of the endoplasmic reticulum (ER) retention signal from the GABAB receptor to enable controlled cell surface expression and signaling by defined metabotropic glutamate receptor (mGluR) heterodimers (6). These studies have shown evidence for asymmetric activation of the heterodimer (11, 90). Furthermore, one agonist can activate the dimer, but two agonists are required for full activation (91). In addition, within the same Family C, T1R3 taste receptors are known to form functional heterodimers with either T1R1 or T1R2 in order to respond to a large panel of ligands and to trigger umami and sweet taste sensations respectively (92).
Unfortunately, related approaches with ER retention signals have been unsuccessful in Family A receptors, and it has not been possible to differentiate clearly the role of each subunit in homomeric and heteromeric signaling with coexpressed receptors. However, multiple lines of study do suggest interaction between Family A receptors in a heteromeric functional unit. Thus, for example, ligand binding dissociation kinetics measurements have recently been linked to the GPCR dimerization process (93). In chemokine receptor heteromers, a CCR2 specific drug accelerates the dissociation of a CCR5 or CXCR4 selective drug when the receptors are coexpressed in heterologous cells and in native lymphocytes (94-96). Moreover, although it remains to be proven conclusively, it seems reasonable to infer that bivalent drugs engaging two different receptors, i.e. heteromer-selective compounds, might act simultaneously on two protomers in a heteromer and thereby directly activate downstream heteromer-specific signaling machinery (97-99) raising the possibility of their selective therapeutic potential (100). Although there is evidence of G protein signaling by coexpressed nonfunctional receptor chimeras, this was proposed to occur by transmembrane domain swapping (101), which is unlikely to be universal (102), and researchers have been unable to generate such functional recovery in adrenergic or dopaminergic receptors. Curiously, coexpression of two loss of function glycoprotein hormone receptors (receptors with either agonist binding or the ability to activate G proteins compromised) (103-105) led to function, but among Family A receptors such rescue seems to be limited to glycoprotein hormone receptors, which have very large extracellular N-terminal binding sites. This is similar to the transactivation seen in the Family C GABAB receptor, in which agonist binding to one protomer signals to G protein through the second protomer (6).
Another example of the potential complexity of receptor interactions is the relationship between the delta opiate receptor (DOR) and the D2R. Although there is substantial evidence for synergy and modulation of signaling in vivo by co-application of selective DOR and D2R ligands (106-108), to the inventor's knowledge, there has been no proposal of direct interaction of these receptors. Curiously, D2R and DOR co-exist in vivo in the striatum in the terminals of dopaminergic neurons, in the terminals of corticostriatal neurons, and in post-synaptic medium spiny neurons (109-110).
In view of the foregoing, there is a need for compositions and methods for evaluating, inter alia, GPCR oligomeric, particularly dimeric, signaling via the oligomer. The present invention is directed to meeting this and other needs.
One embodiment of the present invention is a biological reagent. This biological reagent comprises a complex having (a) a first G-protein coupled receptor (GPCR); and (b) a second GPCR linked to a G-protein, the linkage between the second GPCR and the G-protein being of a length, which prevents productive interaction between the G-protein and the second GPCR, wherein the first GPCR and the second GPCR linked to the G-protein alone are incapable of producing a signal when presented with a ligand.
Another embodiment of the present invention is a method of producing a biological reagent. This method comprises the steps of: (a) expressing a first nucleic acid in a cell, the nucleic acid encoding a first GPCR; (b) expressing a second nucleic acid in the cell, the second nucleic acid encoding a fusion protein comprising a second GPCR fused to a G-protein, the G-protein being fused to the second GPCR in such a manner so that it cannot participate in a productive interaction with the second GPCR; and (c) allowing the expressed proteins from steps (a) and (b) to assemble into a complex in the cell membrane, wherein the expressed proteins from steps (a) and (b) alone are incapable of producing a signal when presented with a ligand.
An additional embodiment of the present invention is a method of determining whether a first and second GPCR have affinity for each other such that they form a functional GPCR oligomer. This method comprises (a) producing or providing a first nucleic acid construct encoding a first GPCR; (b) producing or providing a second nucleic acid construct encoding a second GPCR and its associated G-protein as a fusion protein, the G-protein being fused to the second GPCR in such a manner so that it cannot participate in a productive interaction with the second GPCR, wherein the first GPCR and the second GPCR and its associated G-protein alone are incapable of producing a signal when presented with a ligand; (c) co-expressing the first and second nucleic acid constructs in a cell; and (d) determining the presence of a complex comprising the first and second GPCRs.
A further embodiment of the present invention is a method of determining an effect a compound has on a GPCR oligomer. This method comprises (a) contacting a compound with a first cell expressing a GPCR oligomer having (i) a first GPCR; and (ii) a second GPCR fused to a G-protein, wherein the G-protein is fused to the second GPCR in such a manner so that it cannot participate in a productive interaction with the second GPCR, and the first GPCR and the second GPCR fused to the G-protein alone are incapable of producing a signal when presented with a ligand; (b) detecting the presence of a cellular signal resulting from contact between the compound and the GPCR oligomer; and (c) determining an effect the compound has on the GPCR oligomer.
An additional embodiment of the present invention is a method of identifying a compound capable of interacting with a GPCR oligomer. This method comprises (a) providing a cell expressing a biological reagent according to the present invention; (b) contacting the biological reagent with the compound; and (c) determining whether the compound interacts with the GPCR oligomer.
Yet another embodiment of the present invention is a method of identifying a compound having the ability to modulate binding between a GPCR oligomer and its ligand. This method comprises (a) providing a cell expressing a GPCR oligomer comprising: (i) a first GPCR; and (ii) a second GPCR linked to a G-protein, the linkage between the second GPCR and the G-protein being of a length, which prevents productive interaction between the G-protein and the second GPCR, wherein the first GPCR and the second GPCR linked to the G-protein alone are incapable of producing a signal when presented with a ligand; (b) contacting the cell with a test compound in the presence of the ligand; and (c) comparing the ability of the ligand to bind to the GPCR oligomer with the ability of the ligand to bind to the GPCR oligomer under comparable conditions but in the absence of the compound.
A further embodiment of the present invention is a method for evaluating differential G-protein coupling. This method comprises:
Another embodiment of the present invention is a method of identifying a compound having the ability to modulate the activity of a GPCR oligomer. This method comprises:
A further embodiment of the present invention is a method for evaluating differential effects of a compound on the activity of a GPCR oligomer. This method comprises:
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
One embodiment of the present invention is a biological reagent. This biological reagent comprises a complex having (a) a first G-protein coupled receptor (GPCR); and (b) a second GPCR linked to a G-protein. In this embodiment, the linkage between the second GPCR and the G-protein is of a length, which prevents productive interaction between the G-protein and the second GPCR, and the first GPCR and the second GPCR linked to the G-protein alone are incapable of producing a signal when presented with a ligand.
As used herein, a “complex” means an association comprised of two or more polypeptides, such as e.g., GPCRs, which are in close spatial proximity to each other.
As used herein, “G-protein coupled receptor” or “GPCR” means a 7-transmembrane spanning receptor that upon sensing the appropriate molecule, activates signal transduction pathways and, ultimately, cellular responses, via a guanine nucleotide-binding protein (G-protein). “G-protein” means the α subunit of a heterotrimeric protein that binds guanosine diphosphate (GDP) in its inactive state and binds guanosine triphosphate (GTP) upon activation, and in turn, triggers the signal transduction pathway. The other two subunits of the heterotrimeric protein are β and γ. Exemplary signal transduction pathways include the adenylyl cyclase pathway, the phospholipase C, the Na+/H+ exchanger pathway, changes in inositol 1, 4, 5 triphosphate level or calcium level.
GPCRs and G-proteins of the invention may be wild type proteins, mutant proteins, chimeric proteins, or chimeric proteins which include further mutations. G-proteins may be divided into four subfamilies: the Gs subfamily, the Gi/o subfamily, the Gq/11 subfamily, and the G12/13 subfamily. As used herein, a “Gq/11 subfamily protein” means a G-protein that, upon activation, is able to activate phospholipase C. Non-limiting examples of Gq/11 subfamily proteins according to the present invention include Gq, G11, G14, and G15/16. A “Gi/o subfamily protein” means a G-protein that, upon activation, is able to inhibit adenylyl cyclase and regulate ion channels. Non-limiting examples of Gi/o subfamily proteins according to the present invention include Gi1, Gi2, Gi3, Go1, Go2, Go3, Gz, Gt1, Gt2, and Ggust. A “Gs subfamily protein” means a G-protein that, upon activation, is able to stimulate adenylyl cyclase. Non-limiting examples of Gs subfamily proteins according to the present invention include Gs and Golf. A “G12/13 subfamily protein” means a G-protein that, upon activation, is able to activate the Na+/H+ exchanger pathway. Non-limiting examples of G12/13 subfamily proteins according to the present invention include G12 and G13.
Preferably, the first and/or second GPCRs are class A GPCRs. As used herein, “class A GPCRs” mean GPCRs whose sequences are most similar to rhodopsin. They include, for example, 5-Hydroxytryptamine 1A (5HT1A) receptor, 5-Hydroxytryptamine 1B (5HT1B) receptor, 5-Hydroxytryptamine 1D (5HT1D) receptor, 5-Hydroxytryptamine 2A (5HT2A) receptor, 5-Hydroxytryptamine 2C (5HT2C) receptor, 5-Hydroxytryptamine 4 (5HT4) receptor, 5-Hydroxytryptamine 5A (5HT5A) receptor, 5-Hydroxytryptamine 6 (5HT6) receptor, α1A adrenergic receptor, α1b adrenergic receptor, α2a adrenergic receptor, α2b adrenergic receptor, β1 adrenergic receptor, β2 adrenergic receptor, β3 adrenergic receptor, A1 adenosine receptor, A2 adenosine receptor, A3 adenosine receptor, muscarinic acetylcholine 1 (M1) receptor, muscarinic acetylcholine 2 (M2) receptor, muscarinic acetylcholine 3 (M3) receptor, muscarinic acetylcholine 4 (M4) receptor, Melanocortin2 receptor, angiotensin AT1A receptor, angiotensin AT1B receptor, B2 bradykinin receptor, CXCR3, CXCR4, D1 dopamine receptor, D2 dopamine receptor (D2R), D3 dopamine receptor, D4 dopamine receptor, follicle-stimulating hormone receptor (FSHR), gonadotropin-releasing hormone receptor (GRHR), histamine H1 receptor, histamine H2 receptor, lutropin-choriogonadotropic hormone receptor (LSHR), δ opioid receptor 1; κ opioid receptor 1, μ opioid receptor 1, rhodopsin, Oxytocin receptor, P2U purinoreceptor 1, Prostaglandin D2 receptor, Prostaglandin E2 receptor (EP1 subtype), Somatostatin receptor 2, Somatostatin receptor 5 (SSTR5), thyrotropin releasing hormone (TRH) receptor, Vasopressin 1A receptor, chemokine (C-C motif) receptor 5 (CCR5), and cannabinoid receptor 1. More preferably, the GPCR is a D2R.
The first and the second GPCR may be of the same type. For example, both the first and the second GPCR may be D2R. They may also be different. For example, the first GPCR may be a SSTR5, and the second GPCR may be a D2R.
As used herein, “link” or “linked” means to form a connection, for example, by covalent bonding; and “linkage” refers to such a connection. The connection or linkage may be comprised of amino acids, as in the case of fusion proteins, or comprised of chemically modified bonds. “Productive interaction” means actions that result in the triggering of the appropriate signal transduction pathway. “Signal” means any detectable response, for example, changes in cellular levels of certain chemicals, e.g., Ca2+, or proteins. “Ligand” means a molecule that binds to a GPCR. Such a molecule may be a full or partial agonist, antagonist, inverse agonist, or inverse antagonist.
In one aspect of this embodiment, the complex is present in a cell membrane. Preferably, the cell membrane is part of an intact cell.
In another aspect of this embodiment, the second GPCR and the G-protein are linked as a fusion protein. As used herein, a “fusion protein” means a polypeptide in which two or more proteins, whether wild-type, mutated, or truncated, are joined together. The joining may occur via, for example, molecular genetic techniques, wherein the polynucleotide sequences of the proteins are fused by polymerase chain reaction or by restriction sites, as disclosed herein.
Preferably, the second GPCR is linked directly to a G-protein. As used herein, “linked directly” means having no exogenous intervening amino acids between the two proteins being linked such that the end of one protein being linked is immediately followed by the beginning of the other protein.
In the present invention, the second GPCR may be linked to the G-protein through a linker. As used herein, “linker” means one or more exogenous amino acids between the two proteins being linked or having a chemical bond between the two proteins being linked other than a peptide bond. In the present invention, any amino acid or amino acid derivative or non-peptide bond, which is sufficient to link, e.g., a GPCR to a G-protein may be used so long as the linkage between the GPCR and the G-protein is of a length which prevents productive interaction between GPCR and the G-protein fused to it. Preferably, the linker is from 1 to 3 amino acids in length, such as 2 amino acids in length. In the present invention, when a range is recited, all members of the range, including the end points, are intended.
In anther aspect of this embodiment, the first GPCR and/or the second GPCR are Gi/o-coupled GPCRs. As used herein, a “Gi/o-coupled GPCR” means a GPCR that is able to have productive interactions with a Gi/o subfamily protein. Representative, non-limiting examples of Gi/o-coupled GPCRs according to the present invention include 5HT1A receptor, 5HT1B receptor, 5HT1D receptor, 5-5HT5A receptor, α2a adrenergic receptor, α2b adrenergic receptor, A1 adenosine receptor, A3 adenosine receptor, M2 receptor, M4 receptor, CXCR3, CXCR4, D2R, D3 dopamine receptor, D4 dopamine receptor, FSHR, LSHR, δ opioid receptor 1, κ opioid receptor 1, μ opioid receptor 1, Oxytocin receptor, Somatostatin receptor 2, SSTR5, CCR5, and cannabinoid receptor 1.
In an additional aspect of this embodiment, the first GPCR and/or the second GPCR are Gq/11-coupled GPCRs. In the present invention, a “Gq/11-coupled GPCR” means a GPCR that is able to have productive interactions with a Gq/11 subfamily protein. Representative, non-limiting examples of Gq/11-coupled GPCRs according to the present invention include 5HT2A receptor, 5HT2C receptor, α1A adrenergic receptor, α1b adrenergic receptor, M1 receptor, M3 receptor, dopamine D1 receptor, D2R, angiotensin AT1A receptor, angiotensin AT1B receptor, B2 bradykinin receptor, histamine H1 receptor, GRHR, P2U purinoreceptor 1, Prostaglandin E2 receptor (EP1 subtype), TRH receptor, and Vasopressin receptor.
In a further aspect of this embodiment, the G-protein is a Gqi. As used herein, a “Gqi” means a protein that shares sequence similarities with both Gq/11 and Gi/o subfamily proteins such that the Gqi is activated by a Gi/o-coupled GPCR and activates the Gq/11 signal transduction pathway (e.g., activation of phospholipase C and regulation of ion channels). An example of a Gqi according to the present invention is Gqi5, which is a polypeptide consisting of the amino acid sequence of Gq, except that the last 5 amino acids of Gq are replaced by the last 5 amino acids of Gi1, and that the fourth Cys from the C-terminus of Gi1 is changed to Ile, which makes Gqi5 pertussis toxin (PTX) resistant.
In another aspect of this embodiment, the G-protein is a Gq/11 subfamily protein.
In an additional aspect of this embodiment, the second GPCR comprises a cysteine amino acid toward the terminal end of domain H8, which cysteine is palmitylated. As used herein, “domain H8” refers to helix 8 of the second GPCR, an amphiphilic short helix, which follows transmembrane helix 7 of the second GPCR (111). “Palmitylated” means the addition of a palmityl group to e.g., a cysteine residue (112). For example, in the sequence of the human wild type D2R, short isoform (SEQ ID NO: 61), this palmitylated cysteine towards the terminal end of domain H8 is the last residue (amino acid number 414). Preferably, the G-protein is fused directly to the cysteine amino acid toward the terminal end of H8, which preferably is palmitylated.
The inventors have shown that the palmitylated cysteine towards the terminal end of H8 is highly conserved among members of the Class A Family of GPCRs. (See e.g., sequence alignment in
In another aspect, the G-protein is fused to an amino acid that corresponds to a position selected from the group consisting of position 410, 411, 412, 413, 414, 415, 416, 417, and 418 of the human wild type D2R, short isoform (SEQ ID NO: 61) and isoforms, homologs, and orthologs thereof. As used herein, “isoform” means an alternative form of a protein resulting from differential transcription of the relevant gene either from an alternative promoter or an alternate splicing site. “Homolog” means a gene related to a second gene by descent from a common ancestral DNA sequence. “Ortholog” means a gene in a different species that evolved from a common ancestral gene by speciation.
“Corresponds,” with reference to this embodiment, means consistent with, as done by sequence alignment. Multiple sequence alignment methods including pair-wise sequence alignment methods, may be used to determine the position in a GPCR that corresponds to the positions listed above.
Preferably, the G-protein is fused to an amino acid that corresponds to a position selected from the group consisting of position 413, 414, 415, 416, and 417 of SEQ ID NO: 61 and isoforms, homologs, and orthologs thereof, and more preferably, an amino acid that corresponds to position 414 of SEQ ID NO: 61 and isoforms, homologs, and orthologs thereof. Most preferably, the amino acid is cysteine, and if the amino acid is not cysteine, then the amino acid is modified, using well known procedures, to be cysteine prior to fusion of the G-protein.
In an additional aspect of the embodiment, the first GPCR comprises a mutation. In another aspect, the second GPCR comprises a mutation. In a further aspect, both the first and second GPCRs comprise a mutation. In addition, the G-protein coupled to the second GPCR may be mutated with respect to a wild type form. As used herein, “mutation” means an alteration of the wild type gene, including but not limited to, addition, deletion, or substitution of at least one amino acid. Preferably, the mutation is from 1 to 3 single amino acid substitutions. Also preferably, the mutation creates a mutant D2R. This mutant D2R may be SFD80AGqi5 (SEQ ID NO: 11), SFD80A/CAMGqi5 (SEQ ID NO: 12), sMycD80A (SEQ ID NO: 29), SFD114AGqi5 (SEQ ID NO: 9), SFD114A/CAMGqi5 (SEQ ID NO: 10), sMycD114A (SEQ ID NO: 28), SFR132AGqi5 (SEQ ID NO: 16), SF132A/CAMGqi5 (SEQ ID NO: 17), sMycR132A (SEQ ID NO: 32), SFV136DM140EGqi5 (SEQ ID NO: 18), SFV136DM140E/CAMGqi5 (SEQ ID NO: 19), sMycV136DM140E (SEQ ID NO: 33), SFA213-219Gqi5 (SEQ ID NO: 13), SFA 213-219/CAMGqi5 (SEQ ID NO: 14), sMycA 213-219 (SEQ ID NO: 30), SFAAAA(219-222RRKR) Gqi5 (SEQ ID NO: 4), SFD2S AAAA(219-222RRKR)/CAMGqi5 (SEQ ID NO: 5), sMycAAAA(219-222RRKR) (SEQ ID NO: 25), SFAAAA(IYIV212-215)Gqi5 (SEQ ID NO: 20), sMycAAAA(IVIY212-215), SFN393AGqi5 (SEQ ID NO: 15), SFN393A/CAMGqi5 (SEQ ID NO: 8), sMycN393A (SEQ ID NO: 31), SFD24LGqi5 (SEQ ID NO: 1), SFD24L/CAMGqi5 (SEQ ID NO: 2), sMycD24L (SEQ ID NO: 22), SFCAMGqi5 (SEQ ID NO: 7), sMycD24 short (SEQ ID NO: 23), SFD131A/R132A Gqi5 (SEQ ID NO: 6), sMycCAM (SEQ ID NO: 27), SFD2/D4short Gqi5 (SEQ ID NO: 3), sMycD131N (SEQ ID NO: 39), sMycD131A/R132A (SEQ ID NO: 26), sMycD2S D114A/CAM (SEQ ID NO: 35), sMycD2S D114A/D131N (SEQ ID NO: 36), sMycD2S D114A/R132A (SEQ ID NO: 37), sMycD2S D114AN136D/M140E (SEQ ID NO: 38), or sMycD2S Y397F (Y7.53F) (SEQ ID NO: 40).
In yet another aspect of the embodiment, the complex is capable of producing a signal when presented with a ligand.
Another embodiment of the present invention is a method of producing a biological reagent. This method comprises the steps of: (a) expressing a first nucleic acid in a cell, the nucleic acid encoding a first GPCR; (b) expressing a second nucleic acid in the cell, the second nucleic acid encoding a fusion protein comprising a second GPCR fused to a G-protein, the G-protein being fused to the second GPCR in such a manner so that it cannot participate in a productive interaction with the second GPCR; and (c) allowing the expressed proteins from steps (a) and (b) to assemble into a complex in the cell membrane, wherein the expressed proteins from steps (a) and (b) alone are incapable of producing a signal when presented with a ligand.
In one aspect of this embodiment, the method further comprises, prior to step (a), producing a construct comprising the first nucleic acid encoding the first GPCR and the second nucleic acid encoding the fusion protein of the second GPCR and the G-protein, the G-protein being fused to the second GPCR.
As used herein, a “nucleic acid construct” or “construct” means an artificially constructed segment of nucleic acid that is intended to be introduced into a target tissue or cell, via, e.g., transformation or transfection. It may comprise a DNA sequence encoding a protein of interest, that has been subcloned into a vector, and promoters for expression in the organism. An example of such a construct is set forth in more detail in the Examples below.
In another aspect of this embodiment, the method further comprises, prior to step (a): (i) producing a first construct comprising the first nucleic acid encoding the first GPCR; and (ii) producing a second construct comprising the second nucleic acid encoding the fusion protein of the second GPCR and the G-protein.
In a further aspect of this embodiment, the method further comprises isolating a part of the cell membrane comprising the complex. Isolation of the cell membrane may be accomplished as disclosed in the Examples or by any suitable method known in the art.
An additional embodiment of the present invention is a method of determining whether a first and second GPCR have affinity for each other such that they form, or are capable of forming, a functional GPCR oligomer. This method comprises (a) producing or providing a first nucleic acid construct encoding a first GPCR; (b) producing or providing a second nucleic acid construct encoding a second GPCR and its associated G-protein as a fusion protein, the G-protein being fused to the second GPCR in such a manner so that it cannot participate in a productive interaction with the second GPCR, wherein the first GPCR and the second GPCR and its associated G-protein alone are incapable of producing a signal when presented with a ligand; (c) co-expressing the first and second nucleic acid constructs in a cell; and (d) determining the presence of a complex comprising the first and second GPCRs.
As used herein, “functional” means capable of triggering the appropriate signal transduction pathway upon suitable stimulation. An “oligomer” means dimer, trimer, or an organization of molecules involving even greater numbers of members. In the present embodiment, a dimer—either homodimer or heterodimer—is preferred.
In one aspect of this embodiment, the presence of a complex is determined by contacting the cell with a ligand that binds the first GPCR and determining whether the G-protein is activated. As used herein, an “activated” G-protein is capable of triggering a signaling pathway, resulting in measurable and/or observable changes in levels of molecules, such as calcium levels.
In another aspect, the cell expresses aequorin (AEQ). As used herein, “aequorin” means a photoprotein which emits light upon calcium binding. Such AEQ-expressing cells are described in more detail in the Examples.
Although the present invention is described with reference to AEQ cells, other cell-based systems using different read-outs are contemplated. Especially preferred systems are those that are adapted to high throughput screening (HTS), which, as used herein, defines a process in which large numbers of compounds are tested rapidly and in parallel for binding activity or biological activity against target molecules. In certain embodiments, “large numbers of compounds” may be, for example, more than 100 or more than 300 or more than 500 or more than 1,000 compounds. Preferably, the process is an automated process. HTS is a known method of screening to those skilled in the art.
A further embodiment of the present invention is a method of determining an effect a compound has on a GPCR oligomer. This method comprises (a) contacting a compound with a first cell expressing a GPCR oligomer having (i) a first GPCR; and (ii) a second GPCR fused to a G-protein, wherein the G-protein is fused to the second GPCR in such a manner so that it cannot participate in a productive interaction with the second GPCR, and the first GPCR and the second GPCR fused to the G-protein alone are incapable of producing a signal when presented with a ligand; (b) detecting the presence of a cellular signal resulting from contact between the compound and the GPCR oligomer; and (c) determining an effect the compound has on the GPCR oligomer.
In one aspect of this embodiment, the method further comprises comparing the effect with that resulting from contact between the compound and a mutant of the first GPCR and/or with that resulting from contact between the compound and a mutant of the second GPCR and/or G-protein. Preferably, this method is a HTS.
An additional embodiment of the present invention is a method of identifying a compound capable of interacting with a GPCR oligomer. This method comprises (a) providing a cell expressing a biological reagent according to the present invention; (b) contacting the biological reagent with the compound; and (c) determining whether the compound interacts with the GPCR oligomer.
In one aspect of this embodiment, interaction between the compound and the GPCR oligomer is determined by detecting a change in a cellular signal resulting from the interaction. Preferably, the cellular signal is selected from the group consisting of Ca2+ flux, cAMP levels, inositol 1,4,5 triphosphate levels, protein kinase C activation, and MAP kinase activation.
In another aspect of this embodiment, the cellular signal is determined using a reporter assay. As used herein, “a reporter assay” is a means of detection using a reagent system that detects a change in a cellular signal. Detection of the change may be accomplished through any conventional methodology, including, e.g., radioactive, fluorescent, luminescent, chromogenic, or enzymatic means. For example, one reporter assay, as described herein, utilizes aequorin, which emits blue light upon binding to calcium and thus reflects changes in levels of calcium.
In an additional aspect of this embodiment, the cell further comprises a plasmid encoding apoaequorin and the cellular signal is determined by a change in the luminescence of the cell. Preferably, the cell is a Flp-in T-rex 293 cell.
In a further aspect of this embodiment, the compound interacts with the GPCR oligomer as an agonist, antagonist, inverse agonist, or an inverse antagonist. As used herein, an “agonist” means a substance that binds to a receptor and triggers a response in the cell. An “antagonist” means a substance that does not trigger response itself upon binding to a receptor, but blocks or dampens agonist-mediated responses. An “inverse agonist” is a substance which binds to the same receptor binding-site as an agonist for that receptor and reverses constitutive activity of the receptor. An “inverse antagonist” is a substance which reverses the inverse agonist's activity and restores the receptor's activity.
In yet another aspect of this embodiment, the first GPCR has a modified amino acid sequence compared to the wild-type GPCR sequence so as to render it non-functional. As used herein, “non-functional” means incapable of triggering, or triggering at a substantially reduced rate compared to the wild type GPCR, the appropriate signal transduction pathway upon suitable stimulation. Such modification may be, e.g., a deletion, substitution, or addition of one or more amino acids.
In an additional aspect of this embodiment, the second GPCR is a human D2 receptor (hD2) and the first GPCR is selected from the group consisting of hD1, hD3, hCCR5, hSSTR5, hDOR, hTSHR, hGluR1, hGluR5, hCB1, hA2a, hM4, and h5HT1b. In the present invention, a letter preceding a receptor name refers to its species of origin. Thus, hD2 receptor refers to the human D2 receptor.
In a further aspect of this embodiment, the second GPCR is a mutant D2R as disclosed previously herein.
In another aspect of this embodiment, one of the GPCRs is selected from the group consisting of 3HA-human D1 (SEQ ID NO: 41), 3HAD1-linker-Gqi5 (SEQ ID NO: 42), 3HA-human 5HT1b (SEQ ID NO: 43), 3HA-human A2a (SEQ ID NO: 44), 3HA-human CB1 (SEQ ID NO: 45), mGluR1a (rat) (SEQ ID NO: 46), mGluR5a (rat) (SEQ ID NO: 47), SF-human D3 (SEQ ID NO: 48), SFD3Gqi5 (SEQ ID NO: 49), SFD3-linker-Gqi5 (SEQ ID NO: 50), SF-human SSTR5 (SEQ ID NO: 51), smyc-human SSTR5 (SEQ ID NO: 53), 3HA-M4-linker-Gqi5 (SEQ ID NO: 54), 3HA-M4Gqi5a (SEQ ID NO: 55), human CCR5 (SEQ ID NO: 56), CCR5 Gqi5, (SEQ ID NO: 57), smycDOR (SEQ ID NO: 58), and TSHr Gqi5 (SEQ ID NO: 59).
In an additional aspect of this embodiment, the first GPCR is a wild type D2R and the second GPCR fused to a G protein is D2-Gqi5. Preferably, this method is adapted to be a HTS as set forth previously.
Yet another embodiment of the present invention is a method of identifying a compound having the ability to modulate binding between a GPCR oligomer and its ligand. This method comprises (a) providing a cell expressing a GPCR oligomer comprising: (i) a first GPCR; and (ii) a second GPCR linked to a G-protein, the linkage between the second GPCR and the G-protein being of a length, which prevents productive interaction between the G-protein and the second GPCR, wherein the first GPCR and the second GPCR linked to the G-protein alone are incapable of producing a signal when presented with a ligand; (b) contacting the cell with a test compound in the presence of the ligand; and (c) comparing the ability of the ligand to bind to the GPCR oligomer with the ability of the ligand to bind to the GPCR oligomer under comparable conditions but in the absence of the compound.
As used herein, the ability to “modulate binding” means the ability to change (i.e., increase or decrease) the affinity, in this case, between the GPCR oligomer and its ligand.
In one aspect of this embodiment, the compound is a protein or a peptide. Preferably, the protein is a third GPCR.
In another aspect of this embodiment, the ligand binds to a new or altered ligand binding site determined to be present on the oligomer.
In an additional aspect of this embodiment, the first GPCR, the second GPCR, and/or the G-protein has a modified amino acid sequence compared to a wild-type sequence.
A further embodiment of the present invention is a method for evaluating differential G-protein coupling. This method comprises:
In one aspect of this embodiment, the G-protein is Gqi. In another aspect, the G-protein modulates an intracellular signal selected from the group consisting of Ca2+ level, cAMP level, cGMP level, inositol 1, 4, 5 triphosphate level, diacylglycerol level, protein kinase C activity, and MAP kinase activity. In a further aspect, the first, second, and third cell each express aequorin and the evaluation step comprises detecting luminescence. In this and other aspects of the present invention, endogenous G-proteins may optionally be inactivated with, e.g., PTX or siRNA prior to contacting the cells with a ligand.
Another embodiment of the present invention is a method of identifying a compound having the ability to modulate the activity of a GPCR oligomer. This method comprises:
As used herein, the “activity of a GPCR oligomer” means the amount of productive interactions between the GPCR oligomer and a G-protein. In this context, the ability to “modulate” the activity of a GPCR oligomer means the ability to change (i.e., increase or decrease) the amount of productive interactions between the GPCR oligomer and a G-protein. The amount of productive interaction between a GPCR oligomer and a G-protein may be determined, e.g., by detecting a change in a cellular signal resulting from the interaction, such as Ca2+ flux, cAMP levels, inositol 1,4,5 triphosphate levels, protein kinase C activation, and MAP kinase activation. Cellular signals may be determined by a reporter assay, such as, e.g., those disclosed herein. Suitable cells for use in this method include Flp-in T-rex 293 cells. Preferably, the cell expresses aequorin. This method may be adapted to be a high throughput screen. The compound may interact with the GPCR oligomer as an agonist, antagonist, inverse agonist, or an inverse antagonist. Furthermore, the compound may bind to the ligand binding site of the GPCRs or to an allosteric site.
In one aspect of this embodiment, the compound binds to the second GPCR but not the first GPCR. Alternatively, the compound binds to the first GPCR but not the second GPCR.
In another aspect of this embodiment, the second GPCR is D2R. In a further aspect of this embodiment, the first GPCR is selected from the group consisting of D2R, SSTR5, and DOR.
A further embodiment of the present invention is a method for evaluating differential effects of a compound on the activity of a GPCR oligomer.
This method comprises:
In this embodiment, the activity of the GPCR oligomers may be determined by any means disclosed herein, such as, e.g., a change in a cellular signal resulting from the activity of GPCR oligomers or using any other suitable readout. Preferably, the first and the second cell each express aequorin and the evaluation step comprises detecting luminescence. This method may be adapted to be a high throughput screen. The compound may interact with the GPCR oligomer as an agonist, antagonist, inverse agonist, or an inverse antagonist. Furthermore, the compound may bind to the ligand binding site of the GPCRs or to an allosteric site.
In one aspect of this embodiment, the first, the second, and the fourth GPCRs are the same. In one preferred embodiment, the first, the second, and the fourth GPCRs are D2R. In another preferred embodiment, the third GPCR is SSTR5.
The following examples are provided to further illustrate the compositions and methods of the present invention. These examples are illustrative only and are not intended to limit the scope of the invention in any way.
The D2R agonist quinpirole hydrochloride and the D4R antagonist L745,870 (3-(4-[4-Chlorophenyl]piperazin-1-yl)-methyl-1H-pyrrolo[2,3-b]pyridine trihydrochloride) were from Sigma-Aldrich (St. Louis, Mo.).
Expression plasmids expressing signal peptide flag-tagged short isoform of D2R wild type (114) and mutant receptors were created using standard molecular biology procedures, as described below. Receptor constructs were fused directly through their C-terminus, or through an 8 amino acid linker (FERPADGR, SEQ ID NO: 75), to a PTX-resistant Gqi5. (
The stop codons in the signal peptide flag-tagged D2R short isoform (D2s) wild type (SEQ ID NO: 62) (115) and mutant receptors were removed by PCR, and the sequence TTCGAA was inserted in place of the D2R stop codon to create a BstBI site. Gaqi5 (referred to as Gqi5) (SEQ ID NO: 67) was constructed by replacing the last 5 amino acids of Gaq with those of Gail, except that the fourth residue from the C-terminus was mutated from Cys to Ile. This mutation rendered Gai pertussis-toxin resistant (17). The sequence TTCGAA was also inserted immediately priority to the start codon of Gqi5. D2R-Gqi5 (schematic illustration shown in
Sequences not provided herein are obtainable from publicly available sources, such as the National Center for Biotechnology Information (NCBI).
Flp-in T-rex 293 cells (Invitrogen, Carlsbad, Calif.) were maintained in DMEM medium (GIBCO, Carlsbad, Calif.) supplemented with 10% (v/v) FBS (Gemini, W. Sacramento, Calif.) and 2 mM L-glutamine (Invitrogen). Cells were transfected with Lipofectamine 2000 (Invitrogen) according to manufacturer's protocol. pCIN4AEQ was transfected into Flp-in T-rex 293 cells (Invitrogen), followed by G418 (Mediatech Inc., Manassas, Va.) selecting. Single colonies were isolated, and a clone was identified in which acetylcholine-induced activation of endogenous muscarinic M1 receptors (which couple to endogenous Gq) resulted in robust luminescence in the presence of coelenterazine h (Byosinth AG, Switzerland) (see Aequorin Assay section below).
This parental aequorin cell line was transfected with unfused Myc-tagged D2R (SEQ ID NO: 24) in pIRESpuro3 followed by puromycin (Sigma-Aldrich) selection. After selection, cells were transfected with Flag-tagged D2R-Gqi5 fusion (SEQ ID NO: 64) in pcDNA5/FRT/TO, followed by hygromycin b (Mediatech) selection. Stable coexpression of unfused D2R (SEQ ID NO: 62) with unfused Gqi5 (SEQ ID NO: 67) was achieved by the same strategy. In the Examples, when pertussis toxin treatment is notes, cells were treated with 100 ng/ml pertussis toxin (Sigma-Aldrich) 16-24 hours prior to harvest.
A functional assay based on luminescence of mitochondrial aequorin following intracellular Ca2+ release was performed (23, 24). Cells were seeded in a 15 cm plate, and grown in antibiotics-free medium for about 48 hours until mid-log phase. Tetracycline (1 μg/ml) was added to the medium for 3-24 hours prior to harvest to induce the expression of D2R in a FRT/TO vector (Invitrogen), e.g., pcDNA5/FRT/TO. Cells were dissociated, and then pelleted by centrifuge at 0.6×g for 3 minutes. After washing once with DMEM-F12 medium (Invitrogen, supplemented with 0.1% BSA), cells were resuspended in the same medium to the final concentration of 5×106 cells/ml in the presence of 5 μM coelenterazine h (Biosynth AG). The cell solution was further diluted 10-fold after 4 hours of rotating at room temperature in the dark, followed by one hour incubation under the same conditions. A dose-dependent response was measured by injecting 50 μL cell solution into wells containing 50 μL of different concentrations of an appropriate agonist, such as quinpirole (a D2/D3 receptor agonist), in a 96-well plate. Luminescence signals from the first 15 seconds after injection were read by a POLARstar optima reader (BMG Labtech GmbH, Durham, N.C.). Total response was determined by the signal of injecting 50 μL cell solution into 50 μL assay medium containing 0.1% triton, which raises the Ca++ concentration directly by membrane permeabilization.
The signals were further normalized according to Flag tagged D2R expression level. To normalize for different levels of surface expression levels of the Flag-D2R-Gqi5 mutant constructs, the Emax at each expression level (
Cells that co-expressed Flag tagged D2R Gqi5 fusion and Myc tagged un-fused D2R were induced by 1 μg/ml tetracycline for different amounts of time. An aliquot of the cell solution used for the aequorin assay was used to determine receptor cell surface expression as described in Costagliola et al. (116). Cells were incubated with M2 monoclonal anti-Flag antibody (Sigma) or anti-Myc monoclonal antibody (gift from Cornell) for 30 minutes, followed by another 30 minutes incubation with R-phycoerythrin goat anti mouse IgG (Invitrogen). Cell solutions were diluted to a suitable concentration for FACS assay using Guava Easycyte (Guava technologies, Hayward, Calif.). The surface expression of D2R or D2R-Gqi5 fusion were detected with whole cells without permeation, which could exclude intracellular immature receptors readings.
Cells expressing Flag-D2R-Gqi5 were harvested after induction by tetracycline for varying times from 3 to 24 hours. Cells continuously expressing Myc-D2R were harvested when confluence was suitable. Binding studies were carried out with [3H]N-methylspiperone (PerkinElmer Life Sciences, Waltham, Mass.) using 1 μM sulpiride (Sigma-Aldrich) to define nonspecific binding, as described previously (19). Cells coexpressing D2R or D2R E339A/T343R with free Gqi5 were induced for 20 hours prior to competition binding assay. Intact cells were harvested for binding, and [3H]N-methylspiperone binding was performed as described previously (19).
In the absence of experimentally determined structures of dopamine receptor and Gqi5, the templates for the oligomeric constructs were based on a complex between a heterotrimeric G-protein and rhodopsin. The bovine Gtα subunit was built by homology modeling with MODELLER software (42) from the crystal structure of the complex of a Gtα/Giα chimera and the Gtβγ subunits (PDBID: 1GOT) (41). As the very important C-terminal residues of Gtα (residues 340-350) were missing from the resulting complex, the “activated peptide” of Gt (PDBID: 1 LVZ) was grafted to this structure (43). For this purpose it was necessary to overlap the region Ile340-Glu342 and mutate Ser347 back to Cys. The 1GOT structure of the heterotrimeric Gtαβγ was used to model the Gγβ subunit. As the last residues of Gtγ were missing, the same approach as described above was used to complete the structure: the Gtγ (60-71) farnesyl dodecapeptide (PDBID: 1MF6) (44) in complex with an activated rhodopsin was grafted to the Gt modeled by overlapping Asp60-Asn62. Energy minimization of the Gαβγ was then performed using the AMBER force field (45).
Inspection of the first crystal structure of a heterotrimeric G protein had indicated that the surface area of a GPCR monomer was probably too small to interact simultaneously with both α- and β/γ-subunits of a G protein, leading to the suggestion that the signaling unit could be a dimer. To enable the simultaneous probing of many possible dimer arrangements, an oligomer composed of nine rhodopsin monomers was constructed. The rhodopsin monomers were in the activated form obtained by inclusion of all constrains reported for rhodopsin as reported by Niv et al. (46). Three dimeric interfaces were analyzed: Model 1, in which the dimers have a TM4,5 interface; Model 2, with a symmetric TM4 interface (see Guo et al. (39) for structural details of the interfaces); and Model 3, in which the dimers have a TM1 interface (117).
The docking software used was HADDOCK (High Ambiguity Driven protein-protein DOCKing) (47, 48), which produced one of the best results in the CAPRI (Critical Assessment of PRediction of Interactions) contest and is well characterized in the literature. The docking process for the three models was driven by ambiguous interaction restraints (AIRs) (47) to both monomers. The constraints, which were established from literature-derived experimental data for the binding complex, are presented in Table 2. Notably, the docking protocol of Gt to such models using this set of constraints was verified by the full agreement with the complex obtained for the recent structure of opsin (118) representing a putative activated form of the protein (see below). To select a second protomer for the complex, another docking run was made with restraints only to the central rhodopsin, allowing transducin to explore freely different orientations with respect to the rhodopsin oligomer, and therefore, for the calculation of the relative probabilities of TM1,1 dimers compared to TM4,5 dimer interfaces.
Application of the experimentally-derived constraints took advantage of the distinction made by the HADDOCK algorithm between “active” and “passive” residues. The “active” residues are those considered to be involved in the interaction between the two molecules (Table 3) and to be solvent accessible (either main chain or side chain relative accessibility should be typically >40-50%, which is calculated with the software NACCESS (59)). The “passive” residues are all solvent accessible surface neighbors of active residues. An AIR, the maximum distance between any atom of an active residue of one molecule to any atom of an active or passive residue of the second molecule has a maximum value of 3 Å, as the effective distance deff will always be shorter than the shortest distance entering the sum: deff=[Sum(1/r6)]1/6 In Table 3 below, the distance (in Å) between the Cα of specific residues of rhodopsin and transducin for the two studied models is shown in bold italics. Numbers in brackets are the average distances from the docking solutions from which the optimal representatives were chosen as Model 1 (TM4, 5 dimer) and Model 2 (TM4 dimer) as described above.
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By application of the docking protocol consisting of randomization of orientations and rigid body energy minimization, 1000 different conformations were generated. These structures were ranked according to their average interaction energies (sum of Eelec, Evdw, EAIR). All structures were screened using eighteen restraints given in Table 3. These represent the information extracted from the experimental data and translated into Cα-Cα intermolecular constraints. Set 1 refers to the interactions of the C-terminus of Gα with protomer A (e.g., GPCR1), set 2 to the interactions between the N-terminus of Gα and protomer A, and set 3 to the interactions between the N-terminus of Gα and protomer B (e.g., GPCR2). Sets 1 and 2 in Table 3 contain interactions that could occur simultaneously, whereas set 3 refers to another group of interesting interactions that do not occur simultaneously in the same protomer, for steric reasons. Set 1 was used to filter the most reliable solution because it includes restraints between residues for which experimental support comes from several different sources. In contrast, the interactions of rhodopsin with the N-terminus of Ga are not as well defined. Experimental studies only demonstrate that a broad region of the N-terminus, residues 19 to 28, is involved in the binding interface (40, 53). Kisselev et al. (44) showed that Gγ(50-71), especially Phe64, interacts with the C316 of rhodopsin. Nevertheless, analysis of the rates of Meta II decay led to the proposition that rhodopsin presents two distinct signaling states, one bound to Gtα and one bound to Gtβγ (61).
With this in mind, Gγ was positioned near the binding interface, although not necessarily in direct interaction with rhodopsin. Only Cα-Cα distances<20 Å were interpreted as direct rhodopsin-Gt interactions. A cutoff of 50% fulfillment of the of the interaction criteria was used for accepting valid constructs. The relative probabilities of such valid G protein complexes with the various model dimers (TM4; TM4,5; TM1) were calculated from the corresponding percentages of acceptable complexes found in the resulting set of 1000 structures retrieved from the docking procedure. The construct fulfilling the largest, number of experimentally derived constraints and with the N-terminal helix of Ga parallel to the cytoplasmic face of the rhodopsin dimer, was chosen as the “optimal representative structure” for each model.
The crystal structure of opsin (Ops*) in complex with the GαCT (340-350) segment published recently (27) provided an opportunity for validation of the docking procedure disclosed herein for a cognate monomer in a proposed activated form. Using the computational protocol disclosed herein, the component structures were docked and scored according to the interaction criteria described above. The structures chosen based on these criteria present Root Mean Square Deviation (RMSD) values lower than 2.5 Å in comparison to the crystallographic complex (PDBID: 3DQB) (27), positioning the GαCT ligand in exactly the same binding crevice as observed in the crystal structure. These results confirm the applicability of the procedure and the scoring criteria used to dock the Gt protein.
To enable isolation of the signaling of the D2R from endogenous G proteins, and to control each of the components of the signaling complex, Flp-In 1-Rex-293 cells were engineered to stably express aequorin (AEQ cells) (see Example 1). Aequorin produces luminescence in a calcium-dependent manner in the presence of the substrate coelenterazine (23, 24), and it has been used to create a sensitive luminescence readout for GPCR-mediated PLC activation (25). In these cells, endogenous muscarinic or purinergic receptors signaled robustly via endogenous Gq, resulting in strong agonist-induced (ACH and ATP, respectively) luminescence signals (
To couple D2R activation to a luminescence readout in these cells, a chimeric pertussis toxin-resistant (26) Gq that could signal from Gi-coupled receptors (27) was expressed (See Example 1). D2R signaled robustly when stably co-expressed with this free chimeric Gqi5 or when fused at its C-terminus to Gqi5 through an 8 amino acid linker (D2-linker-Gqi5) (SEQ ID NO: 69) (
Curiously, expression of free Gqi5 (SEQ ID NO: 67) fully rescued the function of the D2-linker-Gqi5G208A (SEQ ID NO: 71) (
To address this problem, another construct was developed. In this construct (D2-Gqi5), Gqi5 was fused more directly to the short cytoplasmic tail of the D2R, via a two amino acid linker. This construct was expressed at the plasma membrane (
Remarkably, however, co-expression in the AEQ cells of D2R and D2-Gqi5, each of which are completely incapable of signaling in assays when expressed alone, led to robust agonist-mediated receptor activation (
In order to manipulate experimentally the function of each protomer, a panel of D2R mutants predicted to be binding and activation-deficient based on findings in the literature for related Class A GPCRs was created and characterized (
When D2/D4 was expressed as protomer A with WT D2R-Gqi5 as protomer B, a reduction in potency and a large decrease in maximal activation by quinpirole was observed (
Interestingly, in the functional complementation assay, the presence of any of the nonbinding or nonsignaling receptor mutants as protomer A completely prevented activation, despite the presence of WT D2R-Gqi5 in protomer B (
In contrast, robust agonist-mediated activation was observed with WT D2R as protomer A and D114A-Gqi5 (SEQ ID NO: 9) (
To explore further the precise arrangement of the signaling unit, mutations were introduced into IL2, which is known to play an important role in coupling to G-proteins. When R1323.50A-Gqi5 (SEQ ID NO: 16) or V1363.54D/M1403.58E-Gqi5 (SEQ ID NO: 18) was expressed as protomer B with wild type D2R as protomer A, no activation was observed. Consistently, the docking studies suggested a critical role for IL2 from both protomers in activating a single G protein (see below). In contrast, these docking studies suggested that only IL3 from protomer A but not that from protomer B is positioned where it can contact the docked G protein. Indeed, experimental results show that the IL3 deletion construct completely wiped out activation when placed in protomer A (
To study the nature of the conformational changes that take place in the transmembrane domains of the dimeric receptor unit, inactivating mutations within the membrane-spanning region was also examined. The transduction-uncoupling mutants D802.50A (myc-tagged version shown in SEQ ID NO: 29), and N7.49393A (myc-tagged version shown in SEQ ID NO: 31), revealed additional differences in the roles of protomers A and B. When either of these mutations was placed in protomer A, signaling was abolished, consistent with the dominant role of this protomer (
As shown in
These data are consistent with the hypothesis that agonist binding to a single protomer maximally activates a signaling unit of two Class A GPCRs and a single G protein, whereas agonist binding to the second protomer inhibits functional response. This presumably reflects the same mechanism by which agonist binding to and activation of the second protomer inhibits signaling. In contrast, findings in the mGluR suggest that although one agonist can activate the dimeric signaling unit, two agonists are required for full activation (38).
It is the active conformation of the second protomer that inhibits signaling, and not agonist binding per se. This is evidenced by the finding that activating protomer B by constitutively activating mutations (
To develop a structural context for this study, independent computational studies that combine molecular modeling with the available experimental data about the modes of interaction of the component GPCRs and G protein in the complexes (but without direct reference to the new findings) were carried out. Because detailed structural information about the D2R is not available, bovine rhodopsin was used as a model for the study. The bovine rhodopsin offers both a known structural template for GPCRs and experimental data about interaction with G protein to guide a protein-protein docking. This experimental data from cross-linking, alanine scanning mutagenesis and other structural and functional studies of the GPCR-G protein interface allowed the identification of several amino acid residues that could be involved in complex formation between both the α- and the βγ-subunits of the G-protein with the respective receptor. The data, derived from the literature, were used not only as constraints to guide transducin docking to a variety of dimer models of rhodopsin (
Both TM41/TM5 and TM1 have been implicated in D2R oligomerization (13, 39, 117). In order to discriminate between a functional dimer with an interface involving TM4 and TM5 (TM4,5 dimer) from one with a TM1 interface (TM1 dimer), the transducin molecular model was docked to a rhodopsin nonamer (
A possible mode of oligomer reorganization associated with function had been suggested based on crosslinking studies in D2R (39) and rhodopsin (40). To evaluate the functional impact of such a reorganization, Gt was docked to the TM4, 5 and TM4 dimer alternatives (
Notably, in the optimal G protein—dimer complex the cytoplasmic ends of TM3 and IL2 from both protomers interact with the docked G protein. This is shown in Model 2 (
Thus, agonist binding to a single protomer maximally activates a signaling unit comprising two Class A GPCRs and a single G protein. Whereas activation of the second protomer inhibits the functional response, inverse agonist binding to the second protomer enhances signaling (
The data and models disclosed herein suggest that the way in which the two protomers contribute to the activated complex with the G protein is not symmetrical, and that activation requires different conformational changes in each protomer. Existing evidence for ligand-induced conformational changes in a second non-binding protomer (11, 12) is consistent with the proposal of conformational changes in both protomers. It has been previously demonstrated an activation-related conformational change at the TM4 dimer interface (39) that also would be consistent with movement of either one or both TM4s. The present finding that transduction-deficient mutants in different TMs differentially affect the ability of protomer B to rescue function is consonant with the importance of conformational changes in this protomer. Interestingly, the apparent negative cooperativity of ligand binding observed in a number of class A GPCRs (93) may well relate to this proposed asymmetry of the signaling unit. For example, in cells expressing chemokine receptor heterodimers, a selective ligand for one protomer leads to dissociation of ligand bound to the other protomer (96), consistent with transmission of an altered conformation across the dimer interface, and with a decreased propensity for simultaneous agonist binding to both protomers.
In summary, the functional complementation assay disclosed herein allows for control of the signaling unit of the human dopamine D2 receptor (D2R) and thus for exploring the individual contributions of each GPCR protomer to G protein signaling. Although a single B2AR or rhodopsin molecule can efficiently activate G protein when reconstituted into a nanodisc, a second protomer is present in vivo and profoundly modulates G protein activation of the first protomer, as shown in the functional complementation studies disclosed herein. Importantly, the studies herein showed that this allosteric modulation of signaling results from a direct interaction of the receptor dimer with the G protein, rather than from a downstream effect. This is likely to explain many of the surprising observations concerning the mutual modulation of heteromeric receptor oligomers by ligand binding to one protomer or the other. Moreover, the studies demonstrate that the constitutive activity of a protomer will modulate the activity of the dimeric signaling unit in which it participates. Thus, inverse agonists at one protomer in a heterodimer are likely to be allosteric potentiators of the signaling of its heterodimer partner, whereas agonists of one protomer will be allosteric inhibitors of the second protomer, offering a mechanistic explanation for the often befuddling observations regarding pharmacological effects of ligands acting on heterodimers. Moreover, the model disclosed herein suggests that modulators might be found that are specific for heterodimers and not homodimers, but heretofore it has not been possible to screen for such compounds without the interference of homodimer-mediated signaling. Indeed, it is possible that findings of functional selectivity, that is, different agonists for a given receptor having different effects on different downstream effectors, might reflect differential pharmacological effects on different heteromeric species (121). The novel methodology disclosed herein makes it possible to identify signaling from a defined heterodimer, and thus to identify modulators of heterodimer function. The modulatory mechanism characterized herein and the approach that made this possible offer a new understanding of GPCR signaling in units composed of at least two GPCRs. Applied to specific systems, the approach will make it possible to understand the effects of drugs that target each protomer of such a signaling unit, either identical or different.
With reference to
Next, fusion constructs will be created in which Gqi5 is placed in frame at the C-terminal end of each putative heteromeric partner and will create a stable line for each in AQ cells (
These lines then will be stably expressed with D2R, and, after confirming surface expression of both constructs, whether quinpirole can signal via the Gqi5 fused to the putative heteromeric partner receptor will be determined (
In parallel, each of the putative heteromeric partners as protomer A will be stably coexpressed together with D2-Gqi5 as protomer B (
Since D2-Gqi5 cannot function on its own but may be activated by a D2R in extremely close proximity, activation of any of the partner receptors by their prototypical agonists will be indicative of signaling through the Gqi5 attached to the D2R and thus signaling through a presumed signaling unit, be it a heterodimer or a higher order complex. Modulatory effects of the D2-agonist, quinpirole, and the inverse agonist, sulpiride, will be tested on the potency and efficacy of signaling via the heteromeric partners (
In all cases, cell surface expression will be monitored by FACS analysis against N-terminal epitopes to insure that the receptors express at the cell surface at comparable levels, and expression will be adjusted when necessary by varying the time after addition of tetracycline. These findings will also be validated in the absence of a fused G protein (
The methodology set forth in Example 4 was carried out with DOR-Gqi5 (SEQ ID NO: 71), at which the delta opiate receptor (DOR) specific agonist DPDPE signals effectively and quinpirole is without effect. Coexpression of D2R leads to robust quinpirole-activation of the Gqi5 fused to mouse DOR. At 5 nM DPDPE, a concentration that produces about 10% activation of DOR-Gqi5, the potency of quinpirole was enhanced 10-fold (
The interactions between somatostatin receptor 5 (SSTR5) and D2R were also examined. Despite being unable to signal directly, D2-Gqi5 (SEQ ID NO. 21) is able to provide G protein to SSTR5 (SEQ ID NO: 97, DNA encoding the myc-tagged version of SSTR5 inserted into the pIRESpuro3 vector is shown in SEQ ID NO: 95), thereby enabling activation by somatostatin (SST). A nonbinding mutant D2R (D114A-Gqi5) (SEQ ID NO. 9) enabled SST activation that was unaffected by dopamine agonist and antagonist. In contrast, WT D2R-Gqi5 enabled SST activation that was subject to profound modulation by dopamine agonists and antagonists. (
These findings suggest that a D2R inverse agonist used as an antipsychotic medication not only blocks D2R signaling but also has the potential to profoundly enhance signaling by D2R heteromeric partners. Moreover enhanced D2R signaling, found for example in the striatum in schizophrenia, is expected to be associated with a reduction in heteromeric partner signaling. This greatly complicates the interpretation of pharmacological data, as different heteromeric partners may play greater or lesser roles in different regions, blurring a simple distinction between “on target” and “off target” effects. Importantly, these findings and the associated methodology allow for screening of compounds that are heteromer selective. Such compounds will exert interprotomer modulation in a particular heteromer but not in a homomer, allowing an unprecedented level of fine tuning of the system.
As preliminary proof of principle, a novel allosteric inhibitor of D2R was studied. This compound binds to an allosteric site in the extracellular loops and leads to a maximal inhibition of D2R signaling of only about 40% (data not shown). Surprisingly, a full shift in the SST curve in the presence of this compound was observed (data not shown), just like what was observed for sulpiride and clozapine, suggesting that a drug with reduced efficacy to inhibit homomeric D2R signaling can still maximally enhance SSTR5 signaling, and suggesting that specificity is possible. An assay may be performed in order to create a platform that can be used to screen for heteromer selective ligands, for example, by screening for compounds that modulate SST signaling in SSTR5-D2 heteromers but not in either homomer.
A stably expressed Gq siRNA cell line will be constructed based on the above-described Flp-in T-Rex 293 (Invitrogen) AEQ cell line in order to knock down endogenous Gq/11. The target sequence for Gq/11 silencing is 5′-GATGTTCGTGGACCTGAAC-3′ (SEQ ID NO: 100) (122, 123). This mature siRNA sequence will be constructed in a pLemiR™ lentiviral vector (Open Biosystem, Huntsville, Ala.), which can be stably expressed in mammalian cells by selecting with puromycin after transfection. Response to activation of endogenous muscarinic receptors will be screened with the goal of achieving sufficient knock down of endogenous Gq/11 to ablate signaling. This will allow for screening for Gq/11-coupled GPCRs co-expressed with GPCR-Gq/11 fusion in this system by a modified FRT/TO vector, which expresses two GPCRs simultaneously. By this means, the interaction between protomers comprised of a Gq/11-coupled GPCR and a GPCR-Gq/11 chimera may be investigated without influence of endogenous Gq/11. Based this special system, the only functional unit will be a heterodimer consisting of an unfused GPCR and a GPCR-Gq/11 chimera. Note that the Gq/11 fusions will be constructed with an altered DNA coding sequence to express a normal protein that is insensitive to the siRNA.
The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.
All patents, patent applications, and documents cited within this application, including those set forth below, are hereby incorporated by reference as if recited in full herein.
Although illustrative embodiments of the present invention have been described herein, it should be understood that the invention is not limited to those described, and that various other changes or modifications may be made by one skilled in the art without departing from the scope or spirit of the invention.
The present application claims benefit to U.S. provisional patent application No. 61/133,714 filed on Jul. 1, 2008, the entire contents of which is incorporated by reference in its entirety as if recited in full herein.
This invention was made with government support under Grant Numbers RO1 MH54137, DA022413, and DA012923 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US09/03933 | 7/1/2009 | WO | 00 | 3/10/2011 |
| Number | Date | Country | |
|---|---|---|---|
| 61133714 | Jul 2008 | US |
