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.     

Chemotype-selective Modes of Action of κ-Opioid Receptor Agonists

The crystal structures of opioid receptors provide a novel platform for inquiry into opioid receptor function. The molecular determinants for activation of the κ-opioid receptor (KOR) were studied using a combination of agonist docking, functional assays, and site-directed mutagenesis. Eighteen positions in the putative agonist binding site of KOR were selected and evaluated for their effects on receptor binding and activation by ligands representing four distinct chemotypes: the peptide dynorphin A(1–17), the arylacetamide U-69593, and the non-charged ligands salvinorin A and the octahydroisoquinolinone carboxamide 1xx. Minimally biased docking of the tested ligands into the antagonist-bound KOR structure generated distinct binding modes, which were then evaluated biochemically and pharmacologically. Our analysis identified two types of mutations: those that affect receptor function primarily via ligand binding and those that primarily affect function. The shared and differential mechanisms of agonist binding and activation in KOR are further discussed. Usually, mutations affecting function more than binding were located at the periphery of the binding site and did not interact strongly with the various ligands. Analysis of the crystal structure along with the present results provide fundamental insights into the activation mechanism of the KOR and suggest that “functional” residues, along with water molecules detected in the crystal structure, may be directly involved in transduction of the agonist binding event into structural changes at the conserved rotamer switches, thus leading to receptor activation.     

Structural basis for hormone recognition by the Human CRFR2{alpha} G protein-coupled receptor

The mammalian corticotropin releasing factor (CRF)/urocortin (Ucn) peptide hormones include four structurally similar peptides, CRF, Ucn1, Ucn2, and Ucn3, that regulate stress responses, metabolism, and cardiovascular function by activating either of two related class B G protein-coupled receptors, CRFR1 and CRFR2. CRF and Ucn1 activate both receptors, whereas Ucn2 and Ucn3 are CRFR2-selective. The molecular basis for selectivity is unclear. Here, we show that the purified N-terminal extracellular domains (ECDs) of human CRFR1 and the CRFR2α isoform are sufficient to discriminate the peptides, and we present three crystal structures of the CRFR2α ECD bound to each of the Ucn peptides. The CRFR2α ECD forms the same fold observed for the CRFR1 and mouse CRFR2β ECDs but contains a unique N-terminal α-helix formed by its pseudo signal peptide. The CRFR2α ECD peptide-binding site architecture is similar to that of CRFR1, and binding of the α-helical Ucn peptides closely resembles CRF binding to CRFR1. Comparing the electrostatic surface potentials of the ECDs suggests a charge compatibility mechanism for ligand discrimination involving a single amino acid difference in the receptors (CRFR1 Glu104/CRFR2α Pro-100) at a site proximate to peptide residue 35 (Arg in CRF/Ucn1, Ala in Ucn2/3). CRFR1 Glu-104 acts as a selectivity filter preventing Ucn2/3 binding because the nonpolar Ala-35 is incompatible with the negatively charged Glu-104. The structures explain the mechanisms of ligand recognition and discrimination and provide a molecular template for the rational design of therapeutic agents selectively targeting these receptors.     

Ligand-induced internalization and recycling of the human neuropeptide Y2 receptor is regulated by its carboxyl-terminal tail

Agonist-induced internalization of G protein-coupled receptors plays an important role in signal regulation. The underlying mechanisms of the internalization of the human neuropeptide Y(2) receptor (hY(2)R), as well as its desensitization, endocytosis, and resensitization are mainly unknown. In the present study we have investigated the role of carboxyl-terminal (C-terminal) Ser/Thr residues and acidic amino acids in regulating receptor internalization, arrestin interaction, and recycling by fluorescence microscopy, cell surface enzyme-linked immunosorbent assay, and bioluminescence resonance energy transfer in several cell lines. Strikingly, C-terminal truncation mutants revealed two different internalization motifs. Whereas a distal motif (373)DSXTEXT(379) was found to be the primary regulatory internalization sequence acting in concert with arrestin-3, the proximal motif (347)DXXXSEXSXT(356) promoted ligand-induced internalization in an arrestin-3-independent manner. Moreover, we identified a regulatory sequence located between these internalization motifs ((357)FKAKKNLEVRKN(368)), which serves as an inhibitory element. We found that hY(2)R recycling is also governed by structural determinants within the proximal internalization motif. In conclusion, these results indicate that the hY(2)R C terminus is involved in multiple molecular events that regulate internalization, interaction with arrestin-3, and receptor resensitization. Our findings provide novel insights into complex mechanisms of controlled internalization of hY(2)R, which is likely applicable to other GPCRs.     

Second extracellular loop of human glucagon-like peptide-1 receptor (GLP-1R) has a critical role in GLP-1 peptide binding and receptor activation

The glucagon-like peptide-1 receptor (GLP-1R) is a therapeutically important family B G protein-coupled receptor (GPCR) that is pleiotropically coupled to multiple signaling effectors and, with actions including regulation of insulin biosynthesis and secretion, is one of the key targets in the management of type II diabetes mellitus. However, there is limited understanding of the role of the receptor core in orthosteric ligand binding and biological activity. To assess involvement of the extracellular loop (ECL) 2 in ligand-receptor interactions and receptor activation, we performed alanine scanning mutagenesis of loop residues and assessed the impact on receptor expression and GLP-1(1-36)-NH(2) or GLP-1(7-36)-NH(2) binding and activation of three physiologically relevant signaling pathways as follows: cAMP formation, intracellular Ca(2+) (Ca(2+)(i)) mobilization, and phosphorylation of extracellular signal-regulated kinases 1 and 2 (pERK1/2). Although antagonist peptide binding was unaltered, almost all mutations affected GLP-1 peptide agonist binding and/or coupling efficacy, indicating an important role in receptor activation. However, mutation of several residues displayed distinct pathway responses with respect to wild type receptor, including Arg-299 and Tyr-305, where mutation significantly enhanced both GLP-1(1-36)-NH(2)- and GLP-1(7-36)-NH(2)-mediated signaling bias for pERK1/2. In addition, mutation of Cys-296, Trp-297, Asn-300, Asn-302, and Leu-307 significantly increased GLP-1(7-36)-NH(2)-mediated signaling bias toward pERK1/2. Of all mutants studied, only mutation of Trp-306 to alanine abolished all biological activity. These data suggest a critical role of ECL2 of the GLP-1R in the activation transition(s) of the receptor and the importance of this region in the determination of both GLP-1 peptide- and pathway-specific effects.     

Evidence that the thyroid-stimulating hormone (TSH) receptor transmembrane domain influences kinetics of TSH binding to the receptor ectodomain

Thyroid-stimulating hormone (TSH)-induced reduction in ligand binding affinity (negative cooperativity) requires TSH receptor (TSHR) homodimerization, the latter involving primarily the transmembrane domain (TMD) but with the extracellular domain (ECD) also contributing to this association. To test the role of the TMD in negative cooperativity, we studied the TSHR ECD tethered to the cell surface by a glycosylphosphatidylinositol (GPI) anchor that multimerizes despite the absence of the TMD. Using the infinite ligand dilution approach, we confirmed that TSH increased the rate of dissociation (k(off)) of prebound (125)I-TSH from CHO cells expressing the TSH holoreceptor. Such negative cooperativity did not occur with TSHR ECD-GPI-expressing cells. However, even in the absence of added TSH, (125)I-TSH dissociated much more rapidly from the TSHR ECD-GPI than from the TSH holoreceptor. This phenomenon, suggesting a lower TSH affinity for the former, was surprising because both the TSHR ECD and TSH holoreceptor contain the entire TSH-binding site, and the TSH binding affinities for both receptor forms should, theoretically, be identical. In ligand competition studies, we observed that the TSH binding affinity for the TSHR ECD-GPI was significantly lower than that for the TSH holoreceptor. Further evidence for a difference in ligand binding kinetics for the TSH holoreceptor and TSHR ECD-GPI was obtained upon comparison of the TSH K(d) values for these two receptor forms at 4 °C versus room temperature. Our data provide the first evidence that the wild-type TSHR TMD influences ligand binding affinity for the ECD, possibly by altering the conformation of the closely associated hinge region that contributes to the TSH-binding site.     

Structural Determinants for the Interaction of Formyl Peptide Receptor 2 with Peptide Ligands

Unlike formyl peptide receptor 1 (FPR1), FPR2/ALX (FPR2) interacts with peptides of diverse sequences but has low affinity for the Escherichia coli-derived chemotactic peptide fMet-Leu-Phe (fMLF). Using computer modeling and site-directed mutagenesis, we investigated the structural requirements for FPR2 to interact with formyl peptides of different length and composition. In calcium flux assay, the N-formyl group of these peptides is necessary for activation of both FPR2 and FPR1, whereas the composition of the C-terminal amino acids appears more important for FPR2 than FPR1. FPR2 interacts better with pentapeptides (fMLFII, fMLFIK) than tetrapeptides (fMLFK, fMLFW) and tripeptide (fMLF) but only weakly with peptides carrying negative charges at the C terminus (e.g. fMLFE). In contrast, FPR1 is less sensitive to negative charges at the C terminus. A CXCR4-based homology model of FPR1 and FPR2 suggested that Asp-281^7.32 is crucial for the interaction of FPR2 with certain formyl peptides as its negative charge may be repulsive with the terminal COO- group of fMLF and negatively charged Glu in fMLFE. Asp-281^7.32 might also form a stable interaction with the positively charged Lys in fMLFK. Site-directed mutagenesis was performed to remove the negative charge at position 281 in FPR2. The D281^7.32G mutant showed improved affinity for fMLFE and fMLF and reduced affinity for fMLFK compared with wild type FPR2. These results indicate that different structural determinants are used by FPR1 and FPR2 to interact with formyl peptides.     

By Interacting with the C-terminal Phe of Apelin,Phe255 and Trp259 in Helix VI of the Apelin Receptor Are Critical for Internalization

Apelin is the endogenous ligand of the orphan seven-transmembrane domain (TM) G protein-coupled receptor APJ. Apelin is involved in the regulation of body fluid homeostasis and cardiovascular functions. We previously showed the importance of the C-terminal Phe of apelin 17 (K17F) in the hypotensive activity of this peptide. Here, we show either by deleting the Phe residue (K16P) or by substituting it by an Ala (K17A), that it plays a crucial role in apelin receptor internalization but not in apelin binding or in Gα_i-protein coupling. Then we built a homology three-dimensional model of the human apelin receptor using the cholecystokinin receptor-1 model as a template, and we subsequently docked K17F into the binding site. We visualized a hydrophobic cavity at the bottom of the binding pocket in which the C-terminal Phe of K17F was embedded by Trp¹⁵² in TMIV and Trp²⁵⁹ and Phe²⁵⁵ in TMVI. Using molecular modeling and site-directed mutagenesis studies, we further showed that Phe²⁵⁵ and Trp²⁵⁹ are key residues in triggering receptor internalization without playing a role in apelin binding or in Gα_i-protein coupling. These findings bring new insights into apelin receptor activation and show that Phe²⁵⁵ and Trp²⁵⁹, by interacting with the C-terminal Phe of the pyroglutamyl form of apelin 13 (pE13F) or K17F, are crucial for apelin receptor internalization.     

Heteromultimerization of cannabinoid CB(1) receptor and orexin OX(1) receptor generates a unique complex in which both protomers are regulated by orexin A

Agonist-induced internalization was observed for both inducible and constitutively expressed forms of the cannabinoid CB(1) receptor. These were also internalized by the peptide orexin A, which has no direct affinity for the cannabinoid CB(1) receptor, but only when the orexin OX(1) receptor was co-expressed along with the cannabinoid CB(1) receptor. This effect of orexin A was concentration-dependent and blocked by OX(1) receptor antagonists. Moreover, the ability of orexin A to internalize the CB(1) receptor was also blocked by CB(1) receptor antagonists. Remarkably, orexin A was substantially more potent in producing internalization of the CB(1) receptor than in causing internalization of the bulk OX(1) receptor population, and this was true in cells in which the CB(1) receptor was maintained at a constant level, whereas levels of OX(1) could be varied and vice versa. Both co-immunoprecipitation and cell surface, homogenous time-resolved fluorescence resonance energy transfer based on covalent labeling of N-terminal "SNAP" and "CLIP" tags present in the extracellular N-terminal domain of the receptors confirmed the capacity of these two receptors to heteromultimerize. These studies confirm the capacity of the CB(1) and OX(1) receptors to interact directly and demonstrate that this complex has unique regulatory characteristics. The higher potency of the agonist orexin A to regulate the CB(1)-OX(1) heteromer compared with the OX(1)-OX(1) homomer present in the same cells and the effects of CB(1) receptor antagonists on the function of orexin A suggest an interplay between these two systems that may modulate appetite, feeding, and wakefulness.     

Ligand-mimicking Receptor Variant Discloses Binding and Activation Mode of Prolactin-releasing Peptide

The prolactin-releasing peptide receptor and its bioactive RF-amide peptide (PrRP20) have been investigated to explore the ligand binding mode of peptide G-protein-coupled receptors (GPCRs). By receptor mutagenesis, we identified the conserved aspartate in the upper transmembrane helix 6 (Asp(6.59)) of the receptor as the first position that directly interacts with arginine 19 of the ligand (Arg(19)). Replacement of Asp(6.59) with Arg(19) of PrRP20 led to D6.59R, which turned out to be a constitutively active receptor mutant (CAM). This suggests that the mutated residue at the top of transmembrane helix 6 mimics Arg(19) by interacting with additional binding partners in the receptor. Next, we generated an initial comparative model of this CAM because no ligand docking was required, and we selected the next set of receptor mutants to find the engaged partners of the binding pocket. In an iterative process, we identified two acidic residues and two hydrophobic residues that form the peptide ligand binding pocket. As all residues are localized on top or in the upper part of the transmembrane domains, we clearly can show that the extracellular surface of the receptor is sufficient for full signal transduction for prolactin-releasing peptide, rather than a deep, membrane-embedded binding pocket. This contributes to the knowledge of the binding of peptide ligands to GPCRs and might facilitate the development of GPCR ligands, but it also provides new targeting of CAMs involved in hereditary diseases.     

Mapping human protease-activated receptor 4 (PAR4) homodimer interface to transmembrane helix 4

Thrombin activates platelets by binding and cleaving protease-activated receptors 1 and 4 (PAR1 and PAR4). Because of the importance of PAR4 activation on platelets in humans and mice and emerging roles for PAR4 in other tissues, experiments were done to characterize the interaction between PAR4 homodimers. Bimolecular fluorescence complementation and bioluminescence resonance energy transfer (BRET) were used to examine the PAR4 homodimer interface. In bimolecular fluorescence complementation experiments, PAR4 formed homodimers that were disrupted by unlabeled PAR4 in a concentration-dependent manner, but not by rhodopsin. In BRET experiments, the PAR4 homodimers showed a specific interaction as indicated by a hyperbolic BRET signal in response to increasing PAR4-GFP expression. PAR4 did not interact with rhodopsin in BRET assays. The threshold maximum BRET signal was disrupted in a concentration-dependent manner by unlabeled PAR4. In contrast, rhodopsin was unable to disrupt the BRET signal, indicating that the disruption of the PAR4 homodimer is not due to nonspecific interactions. A panel of rho-PAR4 chimeras and PAR4 point mutants has mapped the dimer interface to hydrophobic residues in transmembrane helix 4. Finally, mutations that disrupted dimer formation had reduced calcium mobilization in response to the PAR4 agonist peptide. These results link the loss of dimer formation to a loss of PAR4 signaling.     

Cell adhesion receptor GPR133 couples to Gs protein

Adhesion G protein-coupled receptors (GPCR), with their very large and complex N termini, are thought to participate in cell-cell and cell-matrix interactions and appear to be highly relevant in several developmental processes. Their intracellular signaling is still poorly understood. Here we demonstrate that GPR133, a member of the adhesion GPCR subfamily, activates the G(s) protein/adenylyl cyclase pathway. The presence of the N terminus and the cleavage at the GPCR proteolysis site are not required for G protein signaling. G(s) protein coupling was verified by Gα(s) knockdown with siRNA, overexpression of Gα(s), co-expression of the chimeric Gq(s4) protein that routes GPR133 activity to the phospholipase C/inositol phosphate pathway, and missense mutation within the transmembrane domain that abolished receptor activity without changing cell surface expression. It is likely that not only GPR133 but also other adhesion GPCR signal via classical receptor/G protein-interaction.     

Structural basis of M3 muscarinic receptor dimer/oligomer formation

Class A G protein-coupled receptors (GPCRs) are known to form dimers and/or oligomeric arrays in vitro and in vivo. These complexes are thought to play important roles in modulating class A GPCR function. Many studies suggest that residues located on the "outer" (lipid-facing) surface of the transmembrane (TM) receptor core are critically involved in the formation of class A receptor dimers (oligomers). However, no clear consensus has emerged regarding the identity of the TM helices or TM subsegments involved in this process. To shed light on this issue, we have used the M(3) muscarinic acetylcholine receptor (M3R), a prototypic class A GPCR, as a model system. Using a comprehensive and unbiased approach, we subjected all outward-facing residues (70 amino acids total) of the TM helical bundle (TM1-7) of the M3R to systematic alanine substitution mutagenesis. We then characterized the resulting mutant receptors in radioligand binding and functional studies and determined their ability to form dimers (oligomers) in bioluminescence resonance energy transfer saturation assays. We found that M3R/M3R interactions are not dependent on the presence of one specific structural motif but involve the outer surfaces of multiple TM subsegments (TM1-5 and -7) located within the central and endofacial portions of the TM receptor core. Moreover, we demonstrated that the outward-facing surfaces of most TM helices play critical roles in proper receptor folding and/or function. Guided by the bioluminescence resonance energy transfer data, molecular modeling studies suggested the existence of multiple dimeric/oligomeric M3R arrangements, which may exist in a dynamic equilibrium. Given the high structural homology found among all class A GPCRs, our results should be of considerable general relevance.     

Molecular basis of secretin docking to its intact receptor using multiple photolabile probes distributed throughout the pharmacophore

The molecular basis of ligand binding and activation of family B G protein-coupled receptors is not yet clear due to the lack of insight into the structure of intact receptors. Although NMR and crystal structures of amino-terminal domains of several family members support consistency in general structural motifs that include a peptide-binding cleft, there are variations in the details of docking of the carboxyl terminus of peptide ligands within this cleft, and there is no information about siting of the amino terminus of these peptides. There are also no empirical data to orient the receptor amino terminus relative to the core helical bundle domain. Here, we prepared a series of five new probes, incorporating photolabile moieties into positions 2, 15, 20, 24, and 25 of full agonist secretin analogues. Each bound specifically to the receptor and covalently labeled single distinct receptor residues. Peptide mapping of labeled wild-type and mutant receptors identified that the position 15, 20, and 25 probes labeled residues within the distal amino terminus of the receptor, whereas the position 24 probe labeled the amino terminus adjacent to TM1. Of note, the position 2 probe labeled a residue within the first extracellular loop of the receptor, a region not previously labeled, providing an important new constraint for docking the amino-terminal region of secretin to its receptor core. These additional experimentally derived constraints help to refine our understanding of the structure of the secretin-intact receptor complex and provide new insights into understanding the molecular mechanism for activation of family B G protein-coupled receptors.     

Amino Acid Derivatives as Bitter Taste Receptor (T2R) Blockers

In humans, the 25 bitter taste receptors (T2Rs) are activated by hundreds of structurally diverse bitter compounds. However, only five antagonists or bitter blockers are known. In this study, using molecular modeling guided site-directed mutagenesis, we elucidated the ligand-binding pocket of T2R4. We found seven amino acids located in the extracellular side of transmembrane 3 (TM3), TM4, extracellular loop 2 (ECL2), and ECL3 to be involved in T2R4 binding to its agonist quinine. ECL2 residues Asn-173 and Thr-174 are essential for quinine binding. Guided by a molecular model of T2R4, a number of amino acid derivatives were screened for their ability to bind to T2R4. These predictions were tested by calcium imaging assays that led to identification of γ-aminobutryic acid (GABA) and Nα,Nα-bis(carboxymethyl)-l-lysine (BCML) as competitive inhibitors of quinine-activated T2R4 with an IC₅₀ of 3.2 ± 0.3 μm and 59 ± 18 nm, respectively. Interestingly, pharmacological characterization using a constitutively active mutant of T2R4 reveals that GABA acts as an antagonist, whereas BCML acts as an inverse agonist on T2R4. Site-directed mutagenesis confirms that the two novel bitter blockers share the same orthosteric site as the agonist quinine. The signature residues Ala-90 and Lys-270 play important roles in interacting with BCML and GABA, respectively. This is the first report to characterize a T2R endogenous antagonist and an inverse agonist. The novel bitter blockers will facilitate physiological studies focused on understanding the roles of T2Rs in extraoral tissues.     

Second extracellular loop of human glucagon-like peptide-1 receptor (GLP-1R) differentially regulates orthosteric but not allosteric agonist binding and function

The glucagon-like peptide-1 receptor (GLP-1R) is a prototypical family B G protein-coupled receptor that exhibits physiologically important pleiotropic coupling and ligand-dependent signal bias. In our accompanying article (Koole, C., Wootten, D., Simms, J., Miller, L. J., Christopoulos, A., and Sexton, P. M. (2012) J. Biol. Chem. 287, 3642-3658), we demonstrate, through alanine-scanning mutagenesis, a key role for extracellular loop (ECL) 2 of the receptor in propagating activation transition mediated by GLP-1 peptides that occurs in a peptide- and pathway-dependent manner for cAMP formation, intracellular (Ca(2+)(i)) mobilization, and phosphorylation of extracellular signal-regulated kinases 1 and 2 (pERK1/2). In this study, we examine the effect of ECL2 mutations on the binding and signaling of the peptide mimetics, exendin-4 and oxyntomodulin, as well as small molecule allosteric agonist 6,7-dichloro-2-methylsulfonyl-3-tert-butylaminoquinoxaline (compound 2). Lys-288, Cys-296, Trp-297, and Asn-300 were globally important for peptide signaling and also had critical roles in governing signal bias of the receptor. Peptide-specific effects on relative efficacy and signal bias were most commonly observed for residues 301-305, although R299A mutation also caused significantly different effects for individual peptides. Met-303 was more important for exendin-4 and oxyntomodulin action than those of GLP-1 peptides. Globally, ECL2 mutation was more detrimental to exendin-4-mediated Ca(2+)i release than GLP-1(7-36)-NH(2), providing additional evidence for subtle differences in receptor activation by these two peptides. Unlike peptide activation of the GLP-1R, ECL2 mutations had only limited impact on compound 2 mediated cAMP and pERK responses, consistent with this ligand having a distinct mechanism for receptor activation. These data suggest a critical role of ECL2 of the GLP-1R in the activation transition of the receptor by peptide agonists.     

Cross-talk between carboxypeptidase M and the kinin B1 receptor mediates a new mode of G protein-coupled receptor signaling

G protein-coupled receptor (GPCR) signaling is affected by formation of GPCR homo- or heterodimers, but GPCR regulation by other cell surface proteins is not well understood. We reported that the kinin B1 receptor (B1R) heterodimerizes with membrane carboxypeptidase M (CPM), facilitating receptor signaling via CPM-mediated conversion of bradykinin or kallidin to des-Arg kinin B1R agonists. Here, we found that a catalytically inactive CPM mutant that still binds substrate (CPM-E264Q) also facilitates efficient B1R signaling by B2 receptor agonists bradykinin or kallidin. This response required co-expression of B1R and CPM-E264Q in the same cell, was disrupted by antibody that dissociates CPM from B1R, and was not found with a CPM-E264Q-B1R fusion protein. An additional mutation that reduced the affinity of CPM for C-terminal Arg and increased the affinity for C-terminal Lys inhibited the B1R response to bradykinin (with C-terminal Arg) but generated a response to Lys(9)-bradykinin. CPM-E264Q-mediated activation of B1Rs by bradykinin resulted in increased intramolecular fluorescence resonance energy transfer (FRET) in a B1R FRET construct, similar to that generated directly by a B1R agonist. In cytokine-treated human lung microvascular endothelial cells, disruption of B1R-CPM heterodimers inhibited B1R-dependent NO production stimulated by bradykinin and blocked the increased endothelial permeability caused by treatment with bradykinin and pyrogallol (a superoxide generator). Thus, CPM and B1Rs on cell membranes form a critical complex that potentiates B1R signaling. Kinin peptide binding to CPM causes a conformational change in the B1R leading to intracellular signaling and reveals a new mode of GPCR activation by a cell surface peptidase.     

Spatial Approximations between Residues 6 and 12 in the Amino-terminal Region of Glucagon-like Peptide 1 and Its Receptor: A REGION CRITICAL FOR BIOLOGICAL ACTIVITY*

Understanding the molecular basis of natural ligand binding and activation of the glucagon-like peptide 1 (GLP1) receptor may facilitate the development of agonist drugs useful for the management of type 2 diabetes mellitus. We previously reported molecular approximations between carboxyl-terminal residues 24 and 35 within GLP1 and its receptor. In this work, we have focused on the amino-terminal region of GLP1, known to be critical for receptor activation. We developed two high-affinity, full agonist photolabile GLP1 probes having sites of covalent attachment in positions 6 and 12 of the 30-residue peptide (GLP1(7–36)). Both probes bound to the receptor specifically and covalently labeled single distinct sites. Chemical and protease cleavage of the labeled receptor identified the juxtamembrane region of its amino-terminal domain as the region of covalent attachment of the position 12 probe, whereas the region of labeling by the position 6 probe was localized to the first extracellular loop. Radiochemical sequencing identified receptor residue Tyr¹⁴⁵, adjacent to the first transmembrane segment, as the site of labeling by the position 12 probe, and receptor residue Tyr²⁰⁵, within the first extracellular loop, as the site of labeling by the position 6 probe. These data provide support for a common mechanism for natural ligand binding and activation of family B G protein-coupled receptors. This region of interaction of peptide amino-terminal domains with the receptor may provide a pocket that can be targeted by small molecule agonists.     

Two Distinct Determinants of Ligand Specificity in T1R1/T1R3 (the Umami Taste Receptor)

Umami taste perception in mammals is mediated by a heteromeric complex of two G-protein-coupled receptors, T1R1 and T1R3. T1R1/T1R3 exhibits species-dependent differences in ligand specificity; human T1R1/T1R3 specifically responds to l-Glu, whereas mouse T1R1/T1R3 responds more strongly to other l-amino acids than to l-Glu. The mechanism underlying this species difference remains unknown. In this study we analyzed chimeric human-mouse receptors and point mutants of T1R1/T1R3 and identified 12 key residues that modulate amino acid recognition in the human- and mouse-type responses in the extracellular Venus flytrap domain of T1R1. Molecular modeling revealed that the residues critical for human-type acidic amino acid recognition were located at the orthosteric ligand binding site. In contrast, all of the key residues for the mouse-type broad response were located at regions outside of both the orthosteric ligand binding site and the allosteric binding site for inosine-5′-monophosphate (IMP), a known natural umami taste enhancer. Site-directed mutagenesis demonstrated that the newly identified key residues for the mouse-type responses modulated receptor activity in a manner distinct from that of the allosteric modulation via IMP. Analyses of multiple point mutants suggested that the combination of two distinct determinants, amino acid selectivity at the orthosteric site and receptor activity modulation at the non-orthosteric sites, may mediate the ligand specificity of T1R1/T1R3. This hypothesis was supported by the results of studies using nonhuman primate T1R1 receptors. A complex molecular mechanism involving changes in the properties of both the orthosteric and non-orthosteric sites of T1R1 underlies the determination of ligand specificity in mammalian T1R1/T1R3.