G protein-coupled receptor signaling via Src kinase induces endogenous human transient receptor potential vanilloid type 6 (TRPV6) channel activation

Ca(2+) homeostasis plays a critical role in a variety of cellular processes. We showed previously that stimulation of the prostate-specific G protein-coupled receptor (PSGR) enhances cytosolic Ca(2+) and inhibits proliferation of prostate cells. Here, we analyzed the signaling mechanisms underlying the PSGR-mediated Ca(2+) increase. Using complementary molecular, biochemical, electrophysiological, and live-cell imaging techniques, we found that endogenous Ca(2+)-selective transient receptor potential vanilloid type 6 (TRPV6) channels are critically involved in the PSGR-induced Ca(2+) signal. Biophysical characterization of the current activated by PSGR stimulation revealed characteristic properties of TRPV6. The molecular identity of the involved channel was confirmed using RNA interference targeting TrpV6. TRPV6-mediated Ca(2+) influx depended on Src kinase activity. Src kinase activation occurred independently of G protein activation, presumably by direct interaction with PSGR. Taken together, we report that endogenous TRPV6 channels are activated downstream of a G protein-coupled receptor and present the first physiological characterization of these channels in situ.     

Identification of the orphan G protein-coupled receptor GPR31 as a receptor for 12-(S)-hydroxyeicosatetraenoic acid

Hydroxy fatty acids are critical lipid mediators involved in various pathophysiologic functions. We cloned and identified GPR31, a plasma membrane orphan G protein-coupled receptor that displays high affinity for the human 12-lipoxygenase-derived product 12-(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE). Thus, GPR31 is named 12-(S)-HETE receptor (12-HETER) in this study. The cloned 12-HETER demonstrated high affinity binding for 12-(S)-[(3)H]HETE (K(d) = 4.8 ± 0.12 nm). Also, 12-(S)-HETE efficiently and selectively stimulated GTPγS coupling in the membranes of 12-HETER-transfected cells (EC(50) = 0.28 ± 1.26 nm). Activating GTPγS coupling with 12-(S)-HETE proved to be both regio- and stereospecific. Also, 12-(S)-HETE/12-HETER interactions lead to activation of ERK1/2, MEK, and NFκB. Moreover, knocking down 12-HRTER specifically inhibited 12-(S)-HETE-stimulated cell invasion. Thus, 12-HETER represents the first identified high affinity receptor for the 12-(S)-HETE hydroxyl fatty acids.     

Roles of phosphorylation-dependent and -independent mechanisms in the regulation of histamine H2 receptor by G protein-coupled receptor kinase 2

It is widely assumed that G protein-coupled receptor kinase 2 (GRK2)-mediated specific inhibition of G protein-coupled receptors (GPCRs) response involves GRK-mediated receptor phosphorylation followed by β-arrestin binding and subsequent uncoupling from the heterotrimeric G protein. It has recently become evident that GRK2-mediated GPCRs regulation also involves phosphorylation-independent mechanisms. In the present study we investigated whether the histamine H2 receptor (H2R), a Gα(s)-coupled GPCR known to be desensitized by GRK2, needs to be phosphorylated for its desensitization and/or internalization and resensitization. For this purpose we evaluated the effect of the phosphorylating-deficient GRK2K220R mutant on H2R signaling in U937, COS7, and HEK293T cells. We found that although this mutant functioned as dominant negative concerning receptor internalization and resensitization, it desensitized H2R signaling in the same degree as the GRK2 wild type. To identify the domains responsible for the kinase-independent receptor desensitization, we co-transfected the receptor with constructions encoding the GRK2 RGS-homology domain (RH) and the RH or the kinase domain fused to the pleckstrin-homology domain. Results demonstrated that the RH domain of GRK2 was sufficient to desensitize the H2R. Moreover, disruption of RGS functions by the use of GRK2D110A/K220R double mutant, although coimmunoprecipitating with the H2R, reversed GRK2K220R-mediated H2R desensitization. Overall, these results indicate that GRK2 induces desensitization of H2R through a phosphorylation-independent and RGS-dependent mechanism and extends the GRK2 RH domain-mediated regulation of GPCRs beyond Gα(q)-coupled receptors. On the other hand, GRK2 kinase activity proved to be necessary for receptor internalization and the resulting resensitization.     

Recognition in the face of diversity: interactions of heterotrimeric G proteins and G protein-coupled receptor (GPCR) kinases with activated GPCRs

G protein-coupled receptors (GPCRs) represent the largest class of integral membrane protein receptors in the human genome. Despite the great diversity of ligands that activate these GPCRs, they interact with a relatively small number of intracellular proteins to induce profound physiological change. Both heterotrimeric G proteins and GPCR kinases are well known for their ability to specifically recognize GPCRs in their active state. Recent structural studies now suggest that heterotrimeric G proteins and GPCR kinases identify activated receptors via a common molecular mechanism despite having completely different folds.     

Structural Basis of G Protein-coupled Receptor-Gi Protein Interaction: FORMATION OF THE CANNABINOID CB2 RECEPTOR-Gi PROTEIN COMPLEX*

In this study, we applied a comprehensive G protein-coupled receptor-Gα_i protein chemical cross-linking strategy to map the cannabinoid receptor subtype 2 (CB₂)- Gα_i interface and then used molecular dynamics simulations to explore the dynamics of complex formation. Three cross-link sites were identified using LC-MS/MS and electrospray ionization-MS/MS as follows: 1) a sulfhydryl cross-link between C3.53(134) in TMH3 and the Gα_i C-terminal i-3 residue Cys-351; 2) a lysine cross-link between K6.35(245) in TMH6 and the Gα_i C-terminal i-5 residue, Lys-349; and 3) a lysine cross-link between K5.64(215) in TMH5 and the Gα_i α₄β₆ loop residue, Lys-317. To investigate the dynamics and nature of the conformational changes involved in CB₂·G_i complex formation, we carried out microsecond-time scale molecular dynamics simulations of the CB₂ R*·Gα_i1β₁γ₂ complex embedded in a 1-palmitoyl-2-oleoyl-phosphatidylcholine bilayer, using cross-linking information as validation. Our results show that although molecular dynamics simulations started with the G protein orientation in the β2-AR*·Gα_sβ₁γ₂ complex crystal structure, the Gα_i1β₁γ₂ protein reoriented itself within 300 ns. Two major changes occurred as follows. 1) The Gα_i1 α5 helix tilt changed due to the outward movement of TMH5 in CB₂ R*. 2) A 25° clockwise rotation of Gα_i1β₁γ₂ underneath CB₂ R* occurred, with rotation ceasing when Pro-139 (IC-2 loop) anchors in a hydrophobic pocket on Gα_i1 (Val-34, Leu-194, Phe-196, Phe-336, Thr-340, Ile-343, and Ile-344). In this complex, all three experimentally identified cross-links can occur. These findings should be relevant for other class A G protein-coupled receptors that couple to G_i proteins.     

Macromolecular Composition Dictates Receptor and G Protein Selectivity of Regulator of G Protein Signaling (RGS) 7 and 9-2 Protein Complexes in Living Cells

Regulator of G protein signaling (RGS) proteins play essential roles in the regulation of signaling via G protein-coupled receptors (GPCRs). With hundreds of GPCRs and dozens of G proteins, it is important to understand how RGS regulates selective GPCR-G protein signaling. In neurons of the striatum, two RGS proteins, RGS7 and RGS9-2, regulate signaling by μ-opioid receptor (MOR) and dopamine D2 receptor (D2R) and are implicated in drug addiction, movement disorders, and nociception. Both proteins form trimeric complexes with the atypical G protein β subunit Gβ5 and a membrane anchor, R7BP. In this study, we examined GTPase-accelerating protein (GAP) activity as well as Gα and GPCR selectivity of RGS7 and RGS9-2 complexes in live cells using a bioluminescence resonance energy transfer-based assay that monitors dissociation of G protein subunits. We showed that RGS9-2/Gβ5 regulated both Gi and Go with a bias toward Go, but RGS7/Gβ5 could serve as a GAP only for Go. Interestingly, R7BP enhanced GAP activity of RGS7 and RGS9-2 toward Go and Gi and enabled RGS7 to regulate Gi signaling. Neither RGS7 nor RGS9-2 had any activity toward Gz, Gs, or Gq in the absence or presence of R7BP. We also observed no effect of GPCRs (MOR and D2R) on the G protein bias of R7 RGS proteins. However, the GAP activity of RGS9-2 showed a strong receptor preference for D2R over MOR. Finally, RGS7 displayed an four times greater GAP activity relative to RGS9-2. These findings illustrate the principles involved in establishing G protein and GPCR selectivity of striatal RGS proteins.     

Identification of Serine 348 on the Apelin Receptor as a Novel Regulatory Phosphorylation Site in Apelin-13-induced G Protein-independent Biased Signaling

Phosphorylation plays vital roles in the regulation of G protein-coupled receptor (GPCR) functions. The apelin and apelin receptor (APJ) system is involved in the regulation of cardiovascular function and central control of body homeostasis. Here, using tandem mass spectrometry, we first identified phosphorylated serine residues in the C terminus of APJ. To determine the role of phosphorylation sites in APJ-mediated G protein-dependent and -independent signaling and function, we induced a mutation in the C-terminal serine residues and examined their effects on the interaction between APJ with G protein or GRK/β-arrestin and their downstream signaling. Mutation of serine 348 led to an elimination of both GRK and β-arrestin recruitment to APJ induced by apelin-13. Moreover, APJ internalization and G protein-independent ERK signaling were also abolished by point mutation at serine 348. In contrast, this mutant at serine residues had no demonstrable impact on apelin-13-induced G protein activation and its intracellular signaling. These findings suggest that mutation of serine 348 resulted in inactive GRK/β-arrestin. However, there was no change in the active G protein thus, APJ conformation was biased. These results provide important information on the molecular interplay and impact of the APJ function, which may be extrapolated to design novel drugs for cardiac hypertrophy based on this biased signal pathway.     

G Protein-coupled Receptor (GPCR) Signaling via Heterotrimeric G Proteins from Endosomes

Some G protein-coupled receptors (GPCRs), in addition to activating heterotrimeric G proteins in the plasma membrane, appear to elicit a “second wave” of G protein activation after ligand-induced internalization. We briefly summarize evidence supporting this view and then discuss what is presently known about the functional significance of GPCR-G protein activation in endosomes. Endosomal activation can shape the cellular response temporally by prolonging its overall duration, and may shape the response spatially by moving the location of intracellular second messenger production relative to effectors.     

Sphingosine 1-Phosphate Receptor 4 Uses HER2 (ERBB2) to Regulate Extracellular Signal Regulated Kinase-1/2 in MDA-MB-453 Breast Cancer Cells

We demonstrate here that the bioactive lipid sphingosine 1-phosphate (S1P) uses sphingosine 1-phosphate receptor 4 (S1P₄) and human epidermal growth factor receptor 2 (HER2) to stimulate the extracellular signal regulated protein kinase 1/2 (ERK-1/2) pathway in MDA-MB-453 cells. This was based on several lines of evidence. First, the S1P stimulation of ERK-1/2 was abolished by JTE013, which we show here is an S1P_2/4 antagonist and reduced by siRNA knockdown of S1P₄. Second, the S1P-stimulated activation of ERK-1/2 was almost completely abolished by a HER2 inhibitor (ErbB2 inhibitor II) and reduced by siRNA knockdown of HER2 expression. Third, phyto-S1P, which is an S1P₄ agonist, stimulated ERK-1/2 activation in an S1P₄- and HER2-dependent manner. Fourth, FTY720 phosphate, which is an agonist at S1P_1,3,4,5 but not S1P₂ stimulated activation of ERK-1/2. Fifth, S1P stimulated the tyrosine phosphorylation of HER2, which was reduced by JTE013. HER2 which is an orphan receptor tyrosine kinase is the preferred dimerization partner of the EGF receptor. However, EGF-stimulated activation of ERK-1/2 was not affected by siRNA knockdown of HER2 or by ErbB2 (epidermal growth factor receptor 2 (or HER2)) inhibitor II in MDA-MB-453 cells. Moreover, S1P-stimulated activation of ERK-1/2 does not require an EGF receptor. Thus, S1P and EGF function in a mutually exclusive manner. In conclusion, the magnitude of the signaling gain on the ERK-1/2 pathway produced in response to S1P can be increased by HER2 in MDA-MB-453 cells. The linkage of S1P with an oncogene suggests that S1P and specifically S1P₄ may have an important role in breast cancer progression.     

Heterotrimeric G Proteins Directly Regulate MMP14/Membrane Type-1 Matrix Metalloprotease: A NOVEL MECHANISM FOR GPCR-EGFR TRANSACTIVATION*

Agonist stimulation of G protein-coupled receptors (GPCRs) can transactivate epidermal growth factor receptors (EGFRs), but the precise mechanisms for this transactivation have not been defined. Key to this process is the protease-mediated “shedding” of membrane-tethered ligands, which then activate EGFRs. The specific proteases and the events involved in GPCR-EGFR transactivation are not fully understood. We have tested the hypothesis that transactivation can occur by a membrane-delimited process: direct increase in the activity of membrane type-1 matrix metalloprotease (MMP14, MT1-MMP) by heterotrimeric G proteins, and in turn, the generation of heparin-binding epidermal growth factor (HB-EGF) and activation of EGFR. Using membranes prepared from adult rat cardiac myocytes and fibroblasts, we found that MMP14 activity is increased by angiotensin II, phenylephrine, GTP, and guanosine 5′-O-[γ-thio]triphosphate (GTPγS). MMP14 activation by GTPγS occurs in a concentration- and time-dependent manner, does not occur in response to GMP or adenosine 5′-[γ-thio]triphosphate (ATPγS), and is not blunted by inhibitors of Src, PKC, phospholipase C (PLC), PI3K, or soluble MMPs. This activation is specific to MMP14 as it is inhibited by a specific MMP14 peptide inhibitor and siRNA knockdown. MMP14 activation by GTPγS is pertussis toxin-sensitive. A role for heterotrimeric G protein βγ subunits was shown by using the Gβγ inhibitor gallein and the direct activation of recombinant MMP14 by purified βγ subunits. GTPγS-stimulated activation of MMP14 also results in membrane release of HB-EGF and the activation of EGFR. These results define a previously unrecognized, membrane-delimited mechanism for EGFR transactivation via direct G protein activation of MMP14 and identify MMP14 as a heterotrimeric G protein-regulated effector.     

Synergistic Ca2+ responses by G{alpha}i- and G{alpha}q-coupled G-protein-coupled receptors require a single PLC{beta} isoform that is sensitive to both G{beta}{gamma} and G{alpha}q

Cross-talk between Gα(i)- and Gα(q)-linked G-protein-coupled receptors yields synergistic Ca(2+) responses in a variety of cell types. Prior studies have shown that synergistic Ca(2+) responses from macrophage G-protein-coupled receptors are primarily dependent on phospholipase Cβ3 (PLCβ3), with a possible contribution of PLCβ2, whereas signaling through PLCβ4 interferes with synergy. We here show that synergy can be induced by the combination of Gβγ and Gα(q) activation of a single PLCβ isoform. Synergy was absent in macrophages lacking both PLCβ2 and PLCβ3, but it was fully reconstituted following transduction with PLCβ3 alone. Mechanisms of PLCβ-mediated synergy were further explored in NIH-3T3 cells, which express little if any PLCβ2. RNAi-mediated knockdown of endogenous PLCβs demonstrated that synergy in these cells was dependent on PLCβ3, but PLCβ1 and PLCβ4 did not contribute, and overexpression of either isoform inhibited Ca(2+) synergy. When synergy was blocked by RNAi of endogenous PLCβ3, it could be reconstituted by expression of either human PLCβ3 or mouse PLCβ2. In contrast, it could not be reconstituted by human PLCβ3 with a mutation of the Y box, which disrupted activation by Gβγ, and it was only partially restored by human PLCβ3 with a mutation of the C terminus, which partly disrupted activation by Gα(q). Thus, both Gβγ and Gα(q) contribute to activation of PLCβ3 in cells for Ca(2+) synergy. We conclude that Ca(2+) synergy between Gα(i)-coupled and Gα(q)-coupled receptors requires the direct action of both Gβγ and Gα(q) on PLCβ and is mediated primarily by PLCβ3, although PLCβ2 is also competent.     

Targeting CB2-GPR55 Receptor Heteromers Modulates Cancer Cell Signaling

The G protein-coupled receptors CB₂ (CB₂R) and GPR55 are overexpressed in cancer cells and human tumors. Because a modulation of GPR55 activity by cannabinoids has been suggested, we analyzed whether this receptor participates in cannabinoid effects on cancer cells. Here we show that CB₂R and GPR55 form heteromers in cancer cells, that these structures possess unique signaling properties, and that modulation of these heteromers can modify the antitumoral activity of cannabinoids in vivo. These findings unveil the existence of previously unknown signaling platforms that help explain the complex behavior of cannabinoids and may constitute new targets for therapeutic intervention in oncology.     

G Protein-coupled Receptor Kinases of the GRK4 Protein Subfamily Phosphorylate Inactive G Protein-coupled Receptors (GPCRs)

G protein-coupled receptor (GPCR) kinases (GRKs) play a key role in homologous desensitization of GPCRs. It is widely assumed that most GRKs selectively phosphorylate only active GPCRs. Here, we show that although this seems to be the case for the GRK2/3 subfamily, GRK5/6 effectively phosphorylate inactive forms of several GPCRs, including β2-adrenergic and M2 muscarinic receptors, which are commonly used as representative models for GPCRs. Agonist-independent GPCR phosphorylation cannot be explained by constitutive activity of the receptor or membrane association of the GRK, suggesting that it is an inherent ability of GRK5/6. Importantly, phosphorylation of the inactive β₂-adrenergic receptor enhanced its interactions with arrestins. Arrestin-3 was able to discriminate between phosphorylation of the same receptor by GRK2 and GRK5, demonstrating preference for the latter. Arrestin recruitment to inactive phosphorylated GPCRs suggests that not only agonist activation but also the complement of GRKs in the cell regulate formation of the arrestin-receptor complex and thereby G protein-independent signaling.     

Raf kinase inhibitor protein (RKIP) dimer formation controls its target switch from Raf1 to G protein-coupled receptor kinase (GRK) 2

Proteins controlling cellular networks have evolved distinct mechanisms to ensure specificity in protein-protein interactions. Raf kinase inhibitor protein (RKIP) is a multifaceted kinase modulator, but it is not well understood how this small protein (21 kDa) can coordinate its diverse signaling functions. Raf1 and G protein-coupled receptor kinase (GRK) 2 are direct interaction partners of RKIP and thus provide the possibility to untangle the mechanism of its target specificity. Here, we identify RKIP dimer formation as an important mechanistic feature in the target switch from Raf1 to GRK2. Co-immunoprecipitation and cross-linking experiments revealed RKIP dimerization upon phosphorylation of RKIP at serine 153 utilizing purified proteins as well as in cells overexpressing RKIP. A functional phosphomimetic RKIP mutant had a high propensity for dimerization and reproduced the switch from Raf1 to GRK2. RKIP dimerization and GRK2 binding, but not Raf1 interaction, were prevented by a peptide comprising amino acids 127-146 of RKIP, which suggests that this region is critical for dimer formation. Furthermore, a dimeric RKIP mutant displayed a higher affinity to GRK2, but a lower affinity to Raf1. Functional analyses of phosphomimetic as well as dimeric RKIP demonstrated that enhanced dimerization of RKIP translates into decreased Raf1 and increased GRK2 inhibition. The detection of RKIP dimers in a complex with GRK2 in murine hearts implies their physiological relevance. These findings represent a novel mechanistic feature how RKIP can discriminate between its different interaction partners and thus advances our understanding how specific inhibition of kinases can be achieved.     

Selectivity and Evolutionary Divergence of Metabotropic Glutamate Receptors for Endogenous Ligands and G Proteins Coupled to Phospholipase C or TRP Channels

To define the upstream and downstream signaling specificities of metabotropic glutamate receptors (mGluR), we have examined the ability of representative mGluR of group I, II, and III to be activated by endogenous amino acids and catalyze activation of G proteins coupled to phospholipase C (PLC), or activation of G_i/o proteins coupled to the ion channel TRPC4β. Fluorescence-based assays have allowed us to observe interactions not previously reported or clearly identified. We have found that the specificity for endogenous amino acids is remarkably stringent. Even at millimolar levels, structurally similar compounds do not elicit significant activation. As reported previously, the clear exception is l-serine-O-phosphate (l-SOP), which strongly activates group III mGluR, especially mGluR4,-6,-8 but not group I or II mGluR. Whereas l-SOP cannot activate mGluR1 or mGluR2, it acts as a weak antagonist for mGluR1 and a potent antagonist for mGluR2, suggesting that co-recognition of l-glutamate and l-SOP arose early in evolution, and was followed later by divergence of group I and group II mGluR versus group III in l-SOP responses. mGluR7 has low affinity and efficacy for activation by both l-glutamate and l-SOP. Molecular docking studies suggested that residue 74 corresponding to lysine in mGluR4 and asparagine in mGluR7 might play a key role, and, indeed, mutagenesis experiments demonstrated that mutating this residue to lysine in mGluR7 enhances the potency of l-SOP. Experiments with pertussis toxin and dominant-negative Gα_i/o proteins revealed that mGluR1 couples strongly to TRPC4β through Gα_i/o, in addition to coupling to PLC through Gα_q/11.     

Structural domains required for Caenorhabditis elegans G protein-coupled receptor kinase 2 (GRK-2) function in vivo

G protein-coupled receptor kinases (GRKs) are key regulators of signal transduction that specifically phosphorylate activated G protein-coupled receptors (GPCRs) to terminate signaling. Biochemical and crystallographic studies have provided great insight into mammalian GRK2/3 interactions and structure. However, despite extensive in vitro characterization, little is known about the in vivo contribution of these described GRK structural domains and interactions to proper GRK function in signal regulation. We took advantage of the disrupted chemosensory behavior characteristic of Caenorhabditis elegans grk-2 mutants to discern the interactions required for proper in vivo Ce-GRK-2 function. Informed by mammalian crystallographic and biochemical data, we introduced amino acid substitutions into the Ce-grk-2 coding sequence that are predicted to selectively disrupt GPCR phosphorylation, Gα(q/11) binding, Gβγ binding, or phospholipid binding. Changing the most amino-terminal residues, which have been shown in mammalian systems to be required specifically for GPCR phosphorylation but not phosphorylation of alternative substrates or recruitment to activated GPCRs, eliminated the ability of Ce-GRK-2 to restore chemosensory signaling. Disrupting interaction between the predicted Ce-GRK-2 amino-terminal α-helix and kinase domain, posited to stabilize GRKs in their active ATP- and GPCR-bound conformation, also eliminated Ce-GRK-2 chemosensory function. Finally, although changing residues within the RH domain, predicted to disrupt interaction with Gα(q/11), did not affect Ce-GRK-2 chemosensory function, disruption of the predicted PH domain-mediated interactions with Gβγ and phospholipids revealed that both contribute to Ce-GRK-2 function in vivo. Combined, we have demonstrated functional roles for broadly conserved GRK2/3 structural domains in the in vivo regulation of organismal behavior.     

PheVI:09 (Phe6.44) as a Sliding Microswitch in Seven-transmembrane (7TM) G Protein-coupled Receptor Activation

In seven-transmembrane (7TM), G protein-coupled receptors, highly conserved residues function as microswitches, which alternate between different conformations and interaction partners in an extended allosteric interface between the transmembrane segments performing the large scale conformational changes upon receptor activation. Computational analysis using x-ray structures of the β₂-adrenergic receptor demonstrated that PheVI:09 (6.44), which in the inactive state is locked between the backbone and two hydrophobic residues in transmembrane (TM)-III, upon activation slides ∼2 Å toward TM-V into a tight pocket generated by five hydrophobic residues protruding from TM-III and TM-V. Of these, the residue in position III:16 (3.40) (often an Ile or Val) appears to function as a barrier or gate for the transition between inactive and active conformation. Mutational analysis showed that PheVI:09 is essential for the constitutive and/or agonist-induced signaling of the ghrelin receptor, GPR119, the β₂-adrenergic receptor, and the neurokinin-1 receptor. Substitution of the residues constituting the hydrophobic pocket between TM-III and TM-V in the ghrelin receptor in four of five positions impaired receptor signaling. In GPR39, representing the 12% of 7TM receptors lacking an aromatic residue at position VI:09, unchanged agonist-induced signaling was observed upon Ala substitution of LeuVI:09 despite reduced cell surface expression of the mutant receptor. It is concluded that PheVI:09 constitutes an aromatic microswitch that stabilizes the active, outward tilted conformation of TM-VI relative to TM-III by sliding into a tight hydrophobic pocket between TM-III and TM-V and that the hydrophobic residue in position III:16 constitutes a gate for this transition.     

Divergent Transducer-specific Molecular Efficacies Generate Biased Agonism at a G Protein-coupled Receptor (GPCR)

The concept of “biased agonism” arises from the recognition that the ability of an agonist to induce a receptor-mediated response (i.e. “efficacy”) can differ across the multiple signal transduction pathways (e.g. G protein and β-arrestin (βarr)) emanating from a single GPCR. Despite the therapeutic promise of biased agonism, the molecular mechanism(s) whereby biased agonists selectively engage signaling pathways remain elusive. This is due in large part to the challenges associated with quantifying ligand efficacy in cells. To address this, we developed a cell-free approach to directly quantify the transducer-specific molecular efficacies of balanced and biased ligands for the angiotensin II type 1 receptor (AT₁R), a prototypic GPCR. Specifically, we defined efficacy in allosteric terms, equating shifts in ligand affinity (i.e. K_Lo/K_Hi) at AT₁R-G_q and AT₁R-βarr2 fusion proteins with their respective molecular efficacies for activating G_q and βarr2. Consistent with ternary complex model predictions, transducer-specific molecular efficacies were strongly correlated with cellular efficacies for activating G_q and βarr2. Subsequent comparisons across transducers revealed that biased AT₁R agonists possess biased molecular efficacies that were in strong agreement with the signaling bias observed in cellular assays. These findings not only represent the first measurements of the thermodynamic driving forces underlying differences in ligand efficacy between transducers but also support a molecular mechanism whereby divergent transducer-specific molecular efficacies generate biased agonism at a GPCR.     

Role of the transmembrane domain 4/extracellular loop 2 junction of the human gonadotropin-releasing hormone receptor in ligand binding and receptor conformational selection

Recent crystal structures of G protein-coupled receptors (GPCRs) show the remarkable structural diversity of extracellular loop 2 (ECL2), implying its potential role in ligand binding and ligand-induced receptor conformational selectivity. Here we have applied molecular modeling and mutagenesis studies to the TM4/ECL2 junction (residues Pro(174(4.59))-Met(180(4.66))) of the human gonadotropin-releasing hormone (GnRH) receptor, which uniquely has one functional type of receptor but two endogenous ligands in humans. We suggest that the above residues assume an α-helical extension of TM4 in which the side chains of Gln(174(4.60)) and Phe(178(4.64)) face toward the central ligand binding pocket to make H-bond and aromatic contacts with pGlu(1) and Trp(3) of both GnRH I and GnRH II, respectively. The interaction between the side chains of Phe(178(4.64)) of the receptor and Trp(3) of the GnRHs was supported by reciprocal mutations of the interacting residues. Interestingly, alanine mutations of Leu(175(4.61)), Ile(177(4.63)), and Met(180(4.66)) decreased mutant receptor affinity for GnRH I but, in contrast, increased affinity for GnRH II. This suggests that these residues make intramolecular or intermolecular contacts with residues of transmembrane (TM) domain 3, TM5, or the phospholipid bilayer, which couple the ligand structure to specific receptor conformational switches. The marked decrease in signaling efficacy of I177A and F178A also indicates that IIe(177(4.63)) and Phe(178(4.64)) are important in stabilizing receptor-active conformations. These findings suggest that the TM4/ECL2 junction is crucial for peptide ligand binding and, consequently, for ligand-induced receptor conformational selection.