Molecular recognition of corticotropin-releasing factor by its G-protein-coupled receptor CRFR1

The bimolecular interaction between corticotropin-releasing factor (CRF), a neuropeptide, and its type 1 receptor (CRFR1), a class B G-protein-coupled receptor (GPCR), is crucial for activation of the hypothalamic-pituitary-adrenal axis in response to stress, and has been a target of intense drug design for the treatment of anxiety, depression, and related disorders. As a class B GPCR, CRFR1 contains an N-terminal extracellular domain (ECD) that provides the primary ligand binding determinants. Here we present three crystal structures of the human CRFR1 ECD, one in a ligand-free form and two in distinct CRF-bound states. The CRFR1 ECD adopts the alpha-beta-betaalpha fold observed for other class B GPCR ECDs, but the N-terminal alpha-helix is significantly shorter and does not contact CRF. CRF adopts a continuous alpha-helix that docks in a hydrophobic surface of the ECD that is distinct from the peptide-binding site of other class B GPCRs, thereby providing a basis for the specificity of ligand recognition between CRFR1 and other class B GPCRs. The binding of CRF is accompanied by clamp-like conformational changes of two loops of the receptor that anchor the CRF C terminus, including the C-terminal amide group. These structural studies provide a molecular framework for understanding peptide binding and specificity by the CRF receptors as well as a template for designing potent and selective CRFR1 antagonists for therapeutic applications.     

Sauvagine cross-links to the second extracellular loop of the corticotropin-releasing factor type 1 receptor

Contact sites between the corticotropin-releasing factor receptor type 1 (CRFR1), the sauvagine (SVG) radioligands [Tyr(0),Gln(1)]SVG ((125)I-YQS) and [Tyr(0),Gln(1), Leu(17)]SVG ((125)I-YQLS) were examined. (125)I-YQLS or (125)I-YQS was cross-linked to CRFR1 using the chemical cross-linker, disuccinimidyl suberate (DSS), which cross-links the epsilon amino groups of lysine residues that have a molecular distance of 11.4 A. DSS specifically and efficiently cross-linked (125)I-YQLS and (125)I-YQS to CRFR1. CRFR1 contains 5 putative extracellular lysine residues (Lys(110), Lys(111), Lys(113), Lys(257), and Lys(262)) that can cross-link to the 4 lysine residues (Lys(16), Lys(22), Lys(25), and Lys(27)) of the radioligands. Identification of the CNBr-cleaved fragments of CRFR1 cross-linked to (125)I-YQLS or (125)I-YQS established that the second extracellular loop of CRFR1 cross-links to Lys(16) of YQS. Additionally, site-directed mutagenesis (changing Lys to Arg in CRFR1 individually and in combination) revealed that Lys(257) in the second extracellular loop of CRFR1 is an important cross-linking site. In conclusion, it was shown that in SVG-bound CRFR1, Lys(257) of CRFR1 lies in close proximity (11.4 A) to Lys(16) of SVG.     

Novel high-affinity photoactivatable antagonists of corticotropin-releasing factor (CRF) photoaffinity labeling studies on CRF receptor, type 1 (CRFR1).

Novel photoactivatable antagonists of human/rat corticotropin-releasing factor (h/rCRF) have been synthesized and characterized. The N-terminal amino acid D-phenylalanine in astressin ?cyclo(30-33) [D-Phe12, Nle21,38, Glu30, Lys33]h/rCRF-(12-41)?, a potent CRF peptide antagonist, was replaced by a phenyldiazirine, the 4-(1-azi-2,2,2-trifluoroethyl)benzoyl (ATB) residue. Additionally, His32 of astressin was substituted by either alanine or tyrosine for specific radioactive labeling with 125I at either His13 or Tyr32, respectively. The photoactivatable CRF antagonists were tested for their ability to displace 125I-labeled Tyr0 ovine CRF ([125I-labeled Tyr0]oCRF) in binding experiments and to inhibit oCRF-stimulated adenylate cyclase activity in human embryonic kidney (HEK) 293 cells, permanently transfected with cDNA coding for rat CRF receptor, type 1 (rCRFR1) or human Y-79 retinoblastoma cells known to carry endogenous functional human CRFR1 (hCRFR1). ATB-cyclo(30-33)[Nle21,38, Glu30, Ala32, Lys33]h/rCRF-(13-41) (compound 1) was found to bind with higher affinity to rat or human CRFR1 when compared with ATB-cyclo(30-33)[Nle21,38, Glu30, Tyr32, Lys33]h/rCRF-(13-41) (compound 2) and exhibited higher inhibition of oCRF-stimulated cAMP accumulation in HEK 293 cells stably transfected with cDNA coding for rCRFR1 (HEK-rCRFR1 cells) or Y-79 cells. A highly glycosylated, 66-kDa protein was identified with SDS/PAGE, when the radioactively iodinated compounds 1 or 2 were covalently linked to rCRFR1. The specificity of the photoactivatable 125I-labeled CRF antagonists was demonstrated with SDS/PAGE by the finding that these analogs could be displaced from the receptor by their corresponding nonlabeled form, but not other unrelated peptides such as vasoactive intestinal peptide. The observed molecular size of the receptor was in agreement with the size of CRFR1 found in rat pituitary (66 kDa), but was significantly larger than the size of CRFR1 found in rat cerebellum and olfactory bulb (53 kDa).     

Comparison of the biologic actions of corticotropin-releasing factor and sauvagine

Corticotropin-releasing factor (CRF), a peptide isolated from ovine hypothalamus, and sauvagine, a peptide isolated from frog skin, share significant structural homology and elicit a number of similar biological responses. CRF is more potent than sauvagine in stimulating pituitary ACTH secretion. Sauvagine, however, is 5–10 times more potent than CRF to act within the brain to increase plasma levels of catecholamines and glucose and to elevate mean arterial pressure. Sauvagine is likewise more potent than CRF to act outside the brain to increase superior mesenteric artery flow and plasma glucose concentrations and to decrease mean arterial pressure. CRF and sauvagine produce important effects representative of biologically active peptides.     

Functional and protein chemical characterization of the N-terminal domain of the rat corticotropin-releasing factor receptor 1

Rat corticotropin-releasing factor receptor 1 (rCRFR1) was produced either in transfected HEK 293 cells as a complex glycosylated protein or in the presence of the mannosidase I inhibitor kifunensine as a high mannose glycosylated protein. The altered glycosylation did not influence the biological function of rCRFR1 as demonstrated by competitive binding of rat urocortin (rUcn) or human/rat corticotropin-releasing factor (h/rCRF) and agonist-induced cAMP accumulation. The low production rate of the N-terminal domain of rCRFR1 (rCRFR1-NT) by transfected HEK 293 cells, was increased by a factor of 100 in the presence of kifunensine. The product, rCRFR1-NT-Kif, bound rUcn specifically (K(D) = 27 nM) and astressin (K(I) = 60 nM). This affinity was 10-fold lower than the affinity of full length rCRFR1. However, it was sufficiently high for rCRFR1-NT-Kif to serve as a model for the N-terminal domain of rCRFR1. With protein fragmentation, Edman degradation, and mass spectrometric analysis, evidence was found for the signal peptide cleavage site C-terminally to Thr(23) and three disulfide bridges between precursor residues 30 and 54, 44 and 87, and 68 and 102. Of all putative N-glycosylation sites in positions 32, 38, 45, 78, 90, and 98, all Asn residues except for Asn(32) were glycosylated to a significant extent. No O-glycosylation was observed.     

Conformation of the transmembrane domain of the epidermal growth factor receptor

The transmembrane domain of growth factor receptors, such as the epidermal growth factor receptor (EGFR) and the relatedc-erbB-2/neu oncogene protein, has been implicated in the process of receptor dimerization and mitogenic signal transduction, and hence in cellular transformation and oncogenesis. Amino acid substitutions in the transmembrane domain of thec-erbB-2/neu protein that cause a transforming effect may exert this effect through a conformational change from a bend conformation to an-helical structure in this region of the protein, but similar amino acid substitutions at homologous positions in the transmembrane domain of the EGFR (e.g., ValGlu at position 627) fail to have a transforming effect. To examine whether this failure may be due to structural effects, we have used conformational energy analysis to determine the preferred three-dimensional structures for the nonapeptide sequence of the transmembrane domain of the EGFR from residues 623–631 with Val or Glu at position 627. The global minimum energy conformations of both nonapeptides were found to be non--helical with bends at positions 624–625 and 627–628. The failure of the ValGlu substitution to produce a conformational change to an-helix in this region may be responsible for its lack of transforming effect. However, the presence of higher energy-helical conformations for the nonapeptide from the normal EGFR may provide an explanation for the presence of a transforming effect from overexpression of the EGFR.     

NMR structural characterization of a minimal peptide antagonist bound to the extracellular domain of the corticotropin-releasing factor1 receptor

Natural peptide agonists of corticotrophin-releasing factor (CRF) receptors bind to the receptor by a two-site mechanism as follows: the carboxyl end of the ligand binds the N-terminal extracellular domain (ECD) of the receptor and the amino portion of the ligand binds the extracellular face of the seven transmembrane region. Recently, peptide antagonists homologous to the 12 C-terminal residues of CRF have been derived, which bind the CRF(1) receptor through an interaction with the ECD. Here we characterized the binding of a minimal 12-residue peptide antagonist while bound to the isolated ECD of the CRF(1) receptor. We have expressed and purified soluble and properly folded ECD independent from the seven-transmembrane region as a thioredoxin fusion protein in Escherichia coli. A model of the peptide antagonist, cyclic corticotrophin-releasing factor residues 30-41 (cCRF(30-41)), was calculated while bound to the recombinant ECD using transferred nuclear Overhauser effect spectroscopy. Although the peptide is unstructured in solution, it adopts an alpha-helical conformation when bound to the ECD. Residues of cCRF(30-41) comprising the binding interface with the ECD were mapped using saturation transfer difference NMR. Two hydrophobic residues (Met(38) and Ile(41)) as well as two amide groups (Asn(34) and the C-terminal amide) on one face of the helix defined the binding epitope of the antagonist. This epitope may be used as a starting point for development of non-peptide antagonists targeting the ECD of this receptor.     

Structural analysis of the transmembrane domain of the epidermal growth factor receptor

The ligand-binding domain of the epidermal growth factor (EGF) receptor is separated from the cytoplasmic protein tyrosine kinase domain by a predicted single transmembrane segment. Antipeptide antibodies prepared against the outer portion of the predicted transmembrane segment confirmed this area was exposed only when cells were treated with permeabilizing agents. To investigate structural requirements for signal transduction by the transmembrane domain, three types of mutant EGF receptor were prepared. The first type was designed to shorten the transmembrane domain, the second to place proline substitutions within this domain, and the third to make amino acid substitutions analogous to those present in the transforming c-erbB2/neu oncoprotein. Mutant human receptors were expressed in null recipient mouse B82L and Chinese hamster ovary cells. All receptors bound EGF and exhibited EGF-stimulated protein tyrosine kinase activity in vivo as assayed using a 125I-labeled monoclonal anti-phosphotyrosine antibody. EGF stimulated growth of cells expressing each mutant receptor with similar dose-response characteristics. In contrast to other growth factor receptors, the transmembrane domain of the EGF receptor is tolerant to a variety of changes which neither mimic EGF action by constitutive activation nor interfere with ligand-induced signal transduction.     

Resiniferatoxin binds to the capsaicin receptor (TRPV1) near the extracellular side of the S4 transmembrane domain

The capsaicin receptor (TRPV1) is a nonselective cation channel that is activated in nociceptors by several painful stimuli, and hence TRPV1 antagonists could represent a novel class of analgesic compounds. Resiniferatoxin (RTX), a potent agonist of TRPV1, and iodoresiniferatoxin (I-RTX), a potent antagonist of TRPV1, both bind with higher affinity to the rat TRPV1 (rTRPV1) than the human (hTRPV1) isoform. To identify the structural features responsible for this difference in affinity, [(3)H]RTX binding to chimeras between hTRPV1 and rTRPV1 was characterized. The "sensor" region within the transmembrane domain (S1-S4) was found to determine [(3)H]RTX binding affinity. All 16 different residues in this region were systematically substituted in hTRPV1 with rTRPV1 residues. A single mutation in the S4 membrane domain of hTRPV1, L547M, caused a 30-fold increase in [(3)H]RTX affinity whereas the inverse mutation in rTRPV1, M547L, caused a 30-fold decrease in affinity for [(3)H]RTX, and several other agonists and antagonists were similarly affected by these mutations. TRPV1 channels with mutations at position 547 were expressed in oocytes, and the relative response to RTX followed a pattern similar to that seen with [(3)H]RTX binding. These data suggest a model where Met-547 in the S4 domain of TRPV1 forms a binding pocket with Tyr-511 in the S3 domain. This model places RTX near the sensor domain thought to move during the gating process and should help to guide further work designed to understand the gating mechanisms of TRPV1 channels based on comparisons between the agonist RTX and the related competitive antagonist I-RTX.     

A soluble form of the first extracellular domain of mouse type 2beta corticotropin-releasing factor receptor reveals differential ligand specificity

The heptahelical receptors for corticotropin-releasing factor (CRF), CRFR1 and CRFR2, display different specificities for CRF family ligands: CRF and urocortin I bind to CRFR1 with high affinity, whereas urocortin II and III bind to this receptor with very low affinities. In contrast, all the urocortins bind with high affinities, and CRF binds with lower affinity to CRFR2. The first extracellular domain (ECD1) of CRFR1 is important for ligand recognition. Here, we characterize a bacterially expressed soluble protein, ECD1-CRFR2beta, corresponding to the ECD1 of mouse CRFR2beta. The K(i) values for binding to ECD1-CRFR2beta are: astressin = 10.7 (5.4-21.1) nm, urocortin I = 6.4 (4.7-8.7) nm, urocortin II = 6.9 (5.8-8.3) nm, CRF = 97 (22-430) nm, urocortin III = sauvagine >200 nm. These affinities are similar to those for binding to a chimeric receptor in which the ECD1 of CRFR2beta replaces the ECD of the type 1B activin receptor (ALK4). The ECD1-CRFR2beta possesses a disulfide arrangement identical to that of the ECD1 of CRFR1, namely Cys(45)-Cys(70), Cys(60)-Cys(103), and Cys(84)-Cys(118). As determined by circular dichroism, ECD1-CRFR2beta undergoes conformational changes upon binding astressin. These data reinforce the importance of the ECD1 of CRF receptors for ligand recognition and raise the interesting possibility that different ligands having similar affinity for the full-length receptor may, nevertheless, have different affinities for microdomains of the receptor.     

Structure of the first transmembrane domain of the neuronal acetylcholine receptor beta2 subunit

The recent cryoelectron microscopy structure of the Torpedo nicotinic acetylcholine receptor (nAChR) at 4-Å resolution shows long helices for all transmembrane (TM) domains. This is in disagreement with several previous reports that the first TM domain of nAChR and other Cys-loop receptors are not entirely helical. In this study, we determined the structure and backbone dynamics of an extended segment encompassing the first TM domain (TM1e) of nAChR β₂ subunit in dodecylphosphocholine micelles, using solution-state NMR and circular dichroism (CD) spectroscopy. Both CD and NMR results show less helicity in TM1e than in Torpedo nAChR structure (Protein Data Bank: 2BG9). The helical ending residues at the C-terminus are the same in the TM1e NMR structure and the Torpedo nAChR structure, but the helical starting residue (I-217) in TM1e is seven residues closer to the C-terminus. Interestingly, the helical starting residue is two residues before the highly conserved P-219, in accordance with the hypothesis that proline causes helical distortions at three residues preceding it. The NMR relaxation measurements show a dynamics pattern consistent with TM1e structure. The substantial nonhelical content adds greater flexibilities to TM1e, thereby implicating a different molecular basis for nAChR function compared to a longer and more rigid helical TM1.     

Synthesis and SAR of 8-arylquinolines as potent corticotropin-releasing factor1 (CRF1) receptor antagonists

A series of 4-substituted 8-aryl-2-methylquinolines 4 was designed and synthesized as highly potent antagonists for the human CRF(1) receptor. This series of compounds displayed parallel SAR to other bicyclic systems such as pyrazolo[1,5-a]pyrimidines, with several compounds possessing low nanomolar binding affinity. In addition to the high potency, the basicity of this 4-aminoquinoline core may offer CRF(1) antagonists with lower lipophilicity.     

Tryptophan scanning mutagenesis of the first transmembrane domain of the innexin Shaking-B(Lethal)

The channel proteins of gap junctions are encoded by two distinct gene families, connexins, which are exclusive to chordates, and innexins/pannexins, which are found throughout the animal kingdom. Although the relationship between the primary structure and function of the vertebrate connexins has been relatively well studied, there are, to our knowledge, no structure-function analyses of invertebrate innexins. In the first such study, we have used tryptophan scanning to probe the first transmembrane domain (M1) of the Drosophila innexin Shaking-B(Lethal), which is a component of rectifying electrical synapses in the Giant Fiber escape neural circuit. Tryptophan was substituted sequentially for 16 amino acids within M1 of Shaking-B(Lethal). Tryptophan insertion at every fourth residue (H27, T31, L35, and S39) disrupted gap junction function. The distribution of these sites is consistent with helical secondary structure and identifies the face of M1 involved in helix-helix interactions. Tryptophan substitution at several sites in M1 altered channel properties in a variety of ways. Changes in sensitivity to transjunctional voltage (Vj) were common and one mutation (S39W) induced sensitivity to transmembrane voltage (Vm). In addition, several mutations induced hemichannel activity. These changes are similar to those observed after substitutions within the transmembrane domains of connexins.     

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.     

Structure and metal ion binding of the first transmembrane domain of DMT1

DMT1 is an integral membrane protein with 12 putative transmembrane domains. As a divalent metal ion transporter, it plays an important role in metal ion homeostasis from bacteria to human. Loss-function mutations at the conserved motif DPGN located within the first transmembrane domain (TMD1) of DMT1 indicate the significance of TMD1 in the biological function of the protein. In the present work, we study the structure, topology and metal ion binding of DMT1-TMD1 peptide by nuclear magnetic resonance using sodium dodecyl sulfate and dodecylphosphocholine micelles as membrane mimics. We find that the peptide forms an α-helix-extended segment-α-helix configuration in which the motif DPGN locates at the central flexible region. The N-terminal part of the peptide is deeply embedded in micelles, while the motif section and the C-terminal part are close to the surface of micelles. The peptide can bind to Mn2+ and Co2+ ions by the side chains of the negatively charged residues in the motif section and the C-terminal part of TMD1. The crucial role of the central flexible region and the C-terminal part of TMD1 in metal ion capture is confirmed by the binding of the N-terminal part truncated TMD1 to metal ions.     

Dimerization of corticotropin-releasing factor receptor type 1 is not coupled to ligand binding

As described previously, receptor dimerization of G protein-coupled receptors may influence signaling, trafficking, and regulation in vivo. Up to now, most studies aiming at the possible role of receptor dimerization in receptor activation and signal transduction are focused on class A GPCRs. In the present work, the dimerization behavior of the corticotropin-releasing factor receptor type 1 (CRF1R), which belongs to class B of GPCRs and plays an important role in coordination of the immune response, stress, and learning behavior, was investigated by using fluorescence resonance energy transfer (FRET). For this purpose, we generated fusion proteins of CRF1R tagged at their C-terminus to a cyan or yellow fluorescent protein, which can be used as a FRET pair. Binding studies verified that the receptor constructs were able to bind their natural ligands in a manner comparable with the wild-type receptor, whereas cAMP accumulation proved the functionality of the constructs. In microscopic studies, a dimerization of the CRF1R was observed, but the addition of either CRF-related agonists or antagonists did not show any dose-related increase of the observed FRET signal, indicating that the dimer-monomer ratio is not changed on addition of ligand.     

Role of the growth hormone (GH) receptor transmembrane domain in receptor predimerization and GH-induced activation

The GH receptor (GHR) mediates GH effects by activating the GHR-associated cytoplasmic tyrosine kinase, Janus kinase 2. Recent studies indicate that GHRs exist as dimers independently of GH binding. Some authors suggest that receptor predimerization is mediated by the transmembrane domain (TMD) and that GH binding initiates signaling by triggering changes in the orientation of the two GHRs within the dimer. In this study, we investigate the role of GHR TMD in GH-independent receptor dimerization and ligand-induced activation. We prepared a GHR mutant, GHR(LDLR), in which the TMD is replaced with the TMD of the human low-density lipoprotein receptor (LDLR). The resultant chimera has a TMD two residues shorter than the native GHR TMD; thus, in addition to possessing a different TMD, the altered GHR(LDLR) TMD helical register may change positions of the GHR extracellular domain (ECD) and intracellular domain relative to the TMD when compared with the wild-type (WT) receptor. When each was coexpressed with an intracellular domain-truncated GHR mutant, GHR(1-274-Myc), both WT GHR and GHR(LDLR) were specifically coprecipitated with GHR(1-274-Myc), indicating that the GHR TMD was not required for GHR heterodimerization with GHR(1-274-Myc). We further examined the contribution of the so-called "dimerization interface," a GHR ECD region that is critical for GH-induced signaling, to receptor predimerization. Coimmunoprecipitation experiments with either WT GHR, a dimerization interface mutant (GHR-H150D), or a control mutant (GHR-T147D) with GHR(1-274-Myc) showed dramatically reduced coprecipitation of GHR-H150D with GHR(1-274-Myc) when compared with WT GHR or GHR-T147K. This result suggests that, in contrast to some recent models, the dimerization interface contributes to GHR predimerization. We also compared WT GHR with GHR(LDLR) and GHR(LDLRDelta4) (a chimera in which the LDLR TMD has an internal deletion of four residues) with regard to response to GH stimulation. Although the chimeras had similar GH dose responses and time courses for signaling as WT GHR, they were markedly less sensitive to inhibition of signaling by a conformation-sensitive GHR ECD monoclonal antibody. Further, the chimeras were much less sensitive to inducible metalloprotease cleavage than was WT GHR, implying that the ECD conformations of the chimera receptors differ from WT GHR. Collectively, our data indicate that the composition and/or length of the TMD affect some aspects of GHR function, but do not affect receptor predimerization or GH-induced GHR activation. Further, they suggest that the GHR ECD-TMD is more flexible than previously thought in terms of the ability to achieve the active conformation in response to GH.     

Spatial structure of the dimeric transmembrane domain of the growth factor receptor ErbB2 presumably corresponding to the receptor active state

Proper lateral dimerization of the transmembrane domains of receptor tyrosine kinases is required for biochemical signal transduction across the plasma membrane. The spatial structure of the dimeric transmembrane domain of the growth factor receptor ErbB2 embedded into lipid bicelles was obtained by solution NMR, followed by molecular dynamics relaxation in an explicit lipid bilayer. ErbB2 transmembrane segments associate in a right-handed alpha-helical bundle through the N-terminal tandem GG4-like motif Thr652-X3-Ser656-X3-Gly660, providing an explanation for the pathogenic power of some oncogenic mutations.     

Scanning mutagenesis of transmembrane domain 3 of the m1 muscarinic acetylcholine receptor

Scanning mutagenesis of transmembrane domain 3 of the M1 muscarinic acetylcholine receptor has revealed a highly-differentiated α-helical structure. Lipid-facing residues are distinguished from a patch of residues which selectively stabilise the ground state of the receptor, and from a band of amino acids extending the full length of the helix, which contribute to the active agonist-receptor-G protein complex. The most important residues are strongly conserved in the GPCR superfamily.     

Functional analysis of transmembrane domain 2 of the M1 muscarinic acetylcholine receptor

Ala substitution scanning mutagenesis has been used to probe the functional role of amino acids in transmembrane (TM) domain 2 of the M1 muscarinic acetylcholine receptor, and of the highly conserved Asn43 in TM1. The mutation of Asn43, Asn61, and Leu64 caused an enhanced ACh affinity phenotype. Interpreted using a rhodopsin-based homology model, these results suggest the presence of a network of specific contacts between this group of residues and Pro415 and Tyr418 in the highly conserved NPXXY motif in TM7 that exhibit a similar mutagenic phenotype. These contacts may be rearranged or broken when ACh binds. D71A, like N414A, was devoid of signaling activity. We suggest that formation of a direct hydrogen bond between the highly conserved side chains of Asp71 and Asn414 may be a critical feature stabilizing the activated state of the M1 receptor. Mutation of Leu67, Ala70, and Ile74 also reduced the signaling efficacy of the ACh-receptor complex. The side chains of these residues are modeled as an extended surface that may help to orient and insulate the proposed hydrogen bond between Asp71 and Asn414. Mutation of Leu72, Gly75, and Met79 in the outer half of TM2 primarily reduced the expression of functional receptor binding sites. These residues may mediate contacts with TM1 and TM7 that are preserved throughout the receptor activation cycle. Thermal inactivation measurements confirmed that a reduction in structural stability followed the mutation of Met79 as well as Asp71.