Signal transduction by the human thyrotropin receptor: studies on the role of individual amino acid residues in the carboxyl terminal region of the third cytoplasmic loop.

We observed previously that the carboxyl-terminal region of the third loop of the TSH receptor (amino acid residues 617-625) is important in signal transduction. To analyze this region in more detail, in the present study we used site-directed mutagenesis to substitute, on an individual basis, the seven amino acids previously mutated as a group. These amino acids are either charged residues or potential phosphorylation sites. Six of the mutant TSH receptors with individual amino acid substitutions bound TSH with high affinity and displayed a cAMP response to TSH stimulation similar to the wild-type TSH receptor. The mutant receptor TSH-R-Gly625 (Arg----Gly) did not transduce a signal, but these results are noninformative because of the loss of high affinity TSH binding. The present data indicate that for each of the six informative amino acid substitutions, the individual residues are not critical for signal transduction. A corollary of this conclusion is that in the important carboxyl-terminal region of the third cytoplasmic loop of the TSH receptor multiple amino acid residues function as a unit.     

A comprehensive structure-function map of the intracellular surface of the human C5a receptor. I. Identification of critical residues

G protein-coupled receptors are one of the largest protein families in nature; however, the mechanisms by which they activate G proteins are still poorly understood. To identify residues on the intracellular face of the human C5a receptor that are involved in G protein activation, we performed a genetic analysis of each of the three intracellular loops and the carboxyl-terminal tail of the receptor. Amino acid substitutions were randomly incorporated into each loop, and functional receptors were identified in yeast. The third intracellular loop contains the largest number of preserved residues (positions resistant to amino acid substitutions), followed by the second loop, the first loop, and lastly the carboxyl terminus. Surprisingly, complete removal of the carboxyl-terminal tail did not impair C5a receptor signaling. When mapped onto a three-dimensional structural model of the inactive state of the C5a receptor, the preserved residues reside on one half of the intracellular surface of the receptor, creating a potential activation face. Together these data provide one of the most comprehensive functional maps of the intracellular surface of any G protein-coupled receptor to date.     

A comprehensive structure-function map of the intracellular surface of the human C5a receptor. II. Elucidation of G protein specificity determinants

Within any given cell many G protein-coupled receptors are expressed in the presence of multiple G proteins, yet most receptors couple to a specific subset of G proteins to elicit their programmed response. Numerous studies demonstrate that the carboxyl-terminal five amino acids of the Galpha subunits are a major determinant of specificity, however the receptor determinants of specificity are less clear. We have used a collection of 133 functional mutants of the C5a receptor obtained in a mutagenesis screen targeting the intracellular loops and the carboxyl terminus (Matsumoto, M. L., Narzinski, K., Kiser, P. D., Nikiforovich, G. V., and Baranski, T. J. (2007) J. Biol. Chem. 282, 3105-3121) to investigate how specificity is encoded. Each mutant, originally selected for its ability to signal through a nearly full-length Galpha(i) in yeast, was tested to see whether it could activate three versions of chimeric Galpha subunits consisting of Gpa1 fused to the carboxyl-terminal five amino acids of Galpha(i), Galpha(q), or Galpha(s) in yeast. Surprisingly the carboxyl-terminal tail of the C5a receptor is the most important specificity determinant in that nearly all mutants in this region showed a gain in coupling to Galpha(q) and/or Galpha(s). More than half of the receptors mutated in the second intracellular loop also demonstrated broadened G protein coupling. Given a lack of selective advantage for this broadened signaling in the initial screen, we propose a model in which the carboxyl-terminal tail acts together with the intracellular loops to generate a specificity filter for receptor-G protein interactions that functions primarily to restrict access of incorrect G proteins to the receptor.     

Identification of two regions within the cytoplasmic domain of the human interferon-gamma receptor required for function.

Functionally active human interferon-gamma (IFN gamma) receptors require the presence of at least two polypeptides: the IFN gamma receptor and an accessory molecule encoded by a gene on human chromosome 21. Here we have used a murine L cell line that stably contains human chromosome 21 (SCC16-5) to determine whether the receptor's cytoplasmic domain is important for receptor function. SCC16-5 stably transfected with the full-length human IFN gamma receptor cDNA bound, internalized, and responded to human IFN gamma. In contrast, SCC16-5 expressing human IFN gamma receptors lacking a cytoplasmic domain bound human IFN gamma but did not internalize or respond to it. Using a family of IFN gamma receptor deletion mutants, two functionally important regions within the intracellular domain were identified: (a) a membrane proximal region (residues 256-303) required for ligand processing and biologic responsiveness and (b) the carboxyl-terminal 39 amino acids (residues 434-472) needed exclusively for biologic responses.     

The amino terminus of the fourth cytoplasmic loop of rhodopsin modulates rhodopsin-transducin interaction

Rhodopsin is a seven-transmembrane helix receptor that binds and catalytically activates the heterotrimeric G protein transducin (G(t)). This interaction involves the cytoplasmic surface of rhodopsin, which comprises four putative loops and the carboxyl-terminal tail. The fourth loop connects the carboxyl end of transmembrane helix 7 with Cys(322) and Cys(323), which are both modified by membrane-inserted palmitoyl groups. Published data on the roles of the fourth loop in the binding and activation of G(t) are contradictory. Here, we attempt to reconcile these conflicts and define a role for the fourth loop in rhodopsin-G(t) interactions. Fluorescence experiments demonstrated that a synthetic peptide corresponding to the fourth loop of rhodopsin inhibited the activation of G(t) by rhodopsin and interacted directly with the alpha subunit of G(t). A series of rhodopsin mutants was prepared in which portions of the fourth loop were replaced with analogous sequences from the beta(2)-adrenergic receptor or the m1 muscarinic receptor. Chimeric receptors in which residues 310-312 were replaced could not efficiently activate G(t). The defect in G(t) interaction in the fourth loop mutants was not affected by preventing palmitoylation of Cys(322) and Cys(323). We suggest that the amino terminus of the fourth loop interacts directly with G(t), particularly with Galpha(t), and with other regions of the intracellular surface of rhodopsin to support G(t) binding.     

Differential determinants for peptide and non-peptidyl ligand binding to the motilin receptor. Critical role of second extracellular loop for peptide binding and action.

The predicted second extracellular loop domain of the motilin receptor is of particular interest because it is a region that is quite distinct from the analogous regions in other family members that are most closely related and because the initial report of the photoaffinity labeling of a domain of this receptor included this region (Coulie, B. J., Matsuura, B., Dong, M., Hadac, E. M., Pinon, D. I., Feighner, S. D., Howard, A. D., and Miller, L. J. (2001) J. Biol. Chem. 276, 35518-35522). In the current work, motilin receptor constructs were prepared that included sequential deletions ranging from single residues to twelve amino acid segments throughout this 67 amino acid domain. Each construct was expressed in COS cells and characterized for motilin radioligand binding and motilin-stimulated intracellular calcium responses. The only segments that had negative impact on motilin binding and biological activity included deletion constructs DeltaCys(235), Delta179-182, and Delta241-246. Cys(235) is likely involved in the highly conserved and functionally important disulfide bond linking the first and second loops of G protein-coupled receptors. Alanine replacements for each of the amino acid residues in the other two segments revealed that the perimembranous residues at both ends of this loop, Val(179) and Leu(245) and Arg(246), were responsible for the negative impact on motilin binding and biological activity. Of note, these mutants responded normally to the non-peptidyl agonist, erythromycin. These data support important functional roles for both amino-terminal and carboxyl-terminal perimembranous regions of the second loop for responses to the natural agonist peptide, while supporting independent determinants for action of a non-peptidyl agonist ligand.     

Synthetic peptides corresponding to residues 551 to 555 and 650 to 653 of the rat testicular follicle-stimulating hormone (FSH) receptor are sufficient for post-receptor modulation of Sertoli cell responsiveness to FSH stimulation

We have recently demonstrated that synthetic peptides corresponding to the third cytoplasmic (3i) loop (residues 533 to 555) and a region in the carboxy-terminal cytoplasmic tail (residues 645 to 653) of the rat testicular follicle-stimulating hormone receptor (FSHR) affected signal transduction in rat testis membranes and cultured rat Sertoli cells. In order to define more precisely the peptide domains involved, we synthesized truncated peptide amides corresponding to FSHR residues 551–555 (KIAKR) and 650–653 (RKSH), respectively. These two regions were chosen since they contained a minimal structural motif present in G protein activator regions of several other G protein-coupled receptors (i.e., B-X-X-B-B or B-B-X-B, B representing a basic amino acid). Neither peptide inhibited binding of FSH to testis membrane receptors. Each peptide significantly reduced FSH-stimulated estradiol biosynthesis by intact cultured rat Sertoli cells. The same results were obtained with streptolysin O-permeabilized Sertoli cells. No effect was noted on forskolin-induced steroidogenesis, indicating that the peptide effects were not due to interaction with adenylyl cyclase. Each peptide amide, however, induced concentration-dependent increases in guanine nucleotide exchange in rat testis membranes. Our results indicate that interaction of FSH receptor with its associated G protein may involve relatively restricted peptide sequences, and include residues 551–555 (KIAKR) in the third cytoplasmic loop, and residues 650–653 (RKSH) in the carboxy-terminal cytoplasmic tail of the FSH receptor.     

The second intracellular loop of metabotropic glutamate receptors recognizes C termini of G-protein alpha-subunits

Heptahelical receptor coupling selectivity to G-proteins is controlled by a large contact area that involves several portions of the receptor and each subunit of the G-protein. In the G-protein alpha subunit, the C-terminal 5 residues, the N terminus, and the alpha N-beta 1 and alpha 4-alpha 5 loops play important roles. On the receptor side, both the second and third (i2 and i3) intracellular loops as well as the C-terminal tail probably contact these different regions of the G-protein. It is now accepted that the C terminus of the alpha subunit binds in a cavity formed by the i2 and i3 loops. Among the various G-protein-coupled receptors (GPCRs), class III receptors that include metabotropic glutamate (mGlu) receptors greatly differ from the rhodopsin-like GPCRs, but the contact zone between these receptors and the G-protein is less understood. The C terminus of the alpha subunit has been shown to play a pivotal role in the selective recognition of class III GPCRs. Indeed, the mGlu2 and mGlu4 and -8 receptors can discriminate between alpha subunits that differ at the level of their C-terminal end only (such as Gqo and Gqz). Here, we examine the role of the i2 loop of mGluRs in the selective recognition of this region of the alpha subunit. To that aim, we analyzed the coupling properties of mGlu2 and mGlu4 or -8 receptors and chimeras containing the i2 loop of the converse receptor to G-protein alpha subunits that only differ by their C termini (Gqo,Gqz, and their point mutants). Our data demonstrate that the central portion of the i2 loop is responsible for the selective recognition of the C-terminal end of the alpha subunit, especially the residue on position -4. These data are consistent with the proposal that the C-terminal end of the G-protein alpha subunit interacts with residues in a cavity formed by the i2 and i3 loops in class III GPCRs, as reported for class I GPCRs.     

Identification of an interaction between the angiotensin II receptor sub-type AT2 and the ErbB3 receptor, a member of the epidermal growth factor receptor family

To identify the proteins that interact and mediate angiotensin II receptor AT2-specific signaling, a random peptide library was screened by yeast-based Two-Hybrid protein-protein interaction assay technique. A peptide that shared significant homology with the amino acids located between the residues Gly-Xaa-Gly-Xaa-Xaa-Gly721 and Lys742, the residues predicted to be important for ATP binding of the ErbB3 and ErbB2 receptors, was identified to be interacting with the AT2 receptor. The interaction between the human ErbB3 receptor and the AT2 receptor was further confirmed using the cytoplasmic domain (amino acids 671-782) of the human ErbB3 receptor. Moreover, an AT2 receptor peptide that spans the amino acids 226-363, (spans the third ICL and carboxy terminal domain) could also interact with the AT2 receptor in a yeast Two-Hybrid protein-protein interaction assay. Studies using mutated and chimeric AT2 receptors showed that replacing the third intracellular loop (ICL) of the AT2 receptor with that of the AT1 abolishes the interaction between the ErbB3 and the AT2 in yeast Two-Hybrid protein-protein interaction assay. Thus the interaction between the AT2 receptor and the ErbB3 receptor seems to require the region spanning the third ICL and carboxy terminus of the AT2 receptor. Since the third ICL of the AT2 receptor is essential for exerting its inhibitory effects on cell growth, possible involvement of this region in the interaction with the cytoplasmic domain of the ErbB3 receptor suggests a novel signaling mechanism for the AT2 receptor mediated inhibition of cell growth. Furthermore, since both the AT2 and the ErbB3 receptors are expressed during fetal development, we propose that the existence of direct interaction between these two receptors may play a role in the regulation of growth during the initial stages of development.     

Extracellular domain chimeras of the TSH and LH/CG receptors reveal the mid-region (amino acids 171-260) to play a vital role in high affinity TSH binding

We constructed a series of TSH-LH/CG receptor chimeras by homologous substitution of relatively small regions of the TSH receptor extracellular domain for the corresponding region of the extracellular domain of the LH/CG receptor. Constructs were stably expressed in Chinese hamster ovary cells. Of the five chimeric receptors, only TSH-LHR-14, which contains mid-region domain C (amino acid residues 171-260) of the extracellular component of the TSH receptor, exhibited TSH binding of relatively high affinity. Consistent with this TSH binding, chimera TSH-LHR-14 was the only one that demonstrated a functional response to TSH stimulation in terms of intracellular cAMP generation. These data indicate that domain C plays a vital role in TSH receptor function.     

Structural basis of beta-adrenergic receptor function

Receptors that mediate their actions by stimulating guanine nucleotide binding regulatory proteins (G proteins) share structural as well as functional similarities. The structural motif characteristic of receptors of this class includes seven hydrophobic putative transmembrane domains linked by hydrophilic loops. Genetic analysis of the beta-adrenergic receptor (beta AR) revealed that the ligand binding domain of this receptor, like that of rhodopsin, involves residues within the hydrophobic core of the protein. On the basis of these studies, a model for ligand binding to the receptor has been developed in which the amino group of an agonist or antagonist is anchored to the receptor through the carboxylate side chain of Asp113 in the third transmembrane helix. Other interactions between specific residues of the receptor and functional groups on the ligand have also been proposed. The interaction between the beta AR and the G protein Gs has been shown to involve an intracellular region that is postulated to form an amphiphilic alpha helix. This region of the beta AR is also critical for sequestration, which accompanies agonist-mediated desensitization, to occur. Structural similarities among G protein-linked receptors suggest that the information gained from the genetic analysis of the beta AR should help define functionally important regions of other receptors of this class.     

Site-directed mutagenesis of the cytoplasmic domains of the human beta 2-adrenergic receptor. Localization of regions involved in G protein-receptor coupling

Numerous plasma membrane-bound receptors are coupled to various effectors via a family of guanine nucleotide regulatory proteins (G proteins). Amino acid sequences of these receptors, deduced from cDNA and genomic clones, indicate the presence of seven transmembrane-spanning domains. Alignment of the available amino acid sequences of these G protein-linked receptors reveals striking homologies in regions predicted to lie near the cytoplasmic surface of the cell membrane. As these areas are likely those which interact with G proteins, we reasoned that systematic introduction of non-native sequence into these highly conserved regions of the human beta 2-adrenergic receptor would allow resolution of loci participating directly in receptor-G protein coupling. Based on this strategy, we constructed 19 mutant receptor species comprising substitutions and deletions of native sequence in the putative cytoplasmic domains of human beta 2-adrenergic receptor. By monitoring ligand binding characteristics and receptor-mediated stimulation of adenylyl cyclase, we have determined that the C-terminal portion of the third cytoplasmic loop and the N-terminal segment of the cytoplasmic tail appear to be critical for productive receptor-coupling to G proteins. In addition, we have implicated two other areas of the receptor that possibly play supportive roles in maintaining proper orientation of the G protein binding site. These comprise the second cytoplasmic loop and a conserved cysteine residue in the cytoplasmic tail.     

Role of lysine187 within the second extracellular loop of the type A cholecystokinin receptor in agonist-induced activation. Use of complementary charge-reversal mutagenesis to define a functionally important interdomain interaction

Activation of guanine nucleotide-binding protein (G protein)-coupled receptors is believed to involve conformational change that exposes a domain for G protein coupling at the cytosolic surface of the helical confluence, although the mechanisms for achieving this are not well understood. This conformational change can be achieved by docking a diverse variety of agonist ligands, known to occur by interacting with different regions of these receptors. In this study, we focus on the importance of a specific basic residue (Lys187) within the second extracellular loop of the receptor for the peptide hormone, cholecystokinin. Alanine-replacement and charge-reversal mutagenesis of this residue showed that it had no effect on the binding of natural peptide and nonpeptidyl ligands of this receptor but markedly interfered with agonist-stimulated signaling. It was demonstrated that this negative effect on biological activity could be eliminated with the truncation of the first 30 residues of the amino-terminal tail of this receptor. Complementary charge-reversal mutagenesis of each of the five conserved acidic residues within this region of the receptor in the presence of the charge-reversed Lys187 revealed that only the Asp5 mutant fully reversed the negative functional impact of the Lys187 charge reversal. Thus, we have demonstrated that a basic residue within the second extracellular loop of the cholecystokinin receptor interacts with a specific acidic residue within the amino terminus of this receptor. This residue-residue interaction is nicely accommodated within a new molecular model of the agonist-occupied cholecystokinin receptor.     

Effects of truncations of the cytoplasmic tail of the luteinizing hormone/chorionic gonadotropin receptor on receptor-mediated hormone internalization.

The LH/CG receptor is a member of the family of G protein-coupled receptors and consists of a large N-terminal extracellular domain (which is responsible for binding hormone) attached to a region that spans the plasma membrane seven times, ending with an intracellularly located C-terminus. Binding of LH or human CG (hCG) to the LH/CG receptor causes a stimulation of adenylyl cyclase, presumably via activation of Gs. The binding of hormone also leads to its subsequent internalization by receptor-mediated endocytosis. In order to investigate the role of the cytoplasmic tail of this receptor in these events, we prepared a series of mutants in which progressively larger portions of the cytoplasmic tail were deleted. Deletion of 58 amino acids from the C-terminus, in which only 11 cytoplasmic residues remain, resulted in a receptor that was not expressed on the plasma membrane. Receptors rat LHR (rLHR)-t653 and rLHR-t631, in which 21 or 43 amino acids were removed, respectively, were properly expressed. These results suggest that a region(s) between residues 616 and 631 of the rLH/CG receptor are required for proper insertion and/or targeting of the receptor into the plasma membrane. Cells expressing rLHR-t653 or rLHR-t631 bound hCG with the same high affinity as cells expressing the full-length receptor, and basal levels of cAMP were the same among the cells. However, cells expressing the truncated receptors responded to hCG with approximately 2-fold greater levels of maximal cAMP accumulation than cells expressing the full-length receptor. Deletion of up to 43 amino acids from the C-terminus of the rLH/CG receptor had no deleterious effect on hCG internalization. In fact, mutants lacking 21 and 43 amino acids exhibited progressively faster rates of hCG internalization as compared to the full-length receptor. Once internalized, hCG was also degraded at a faster rate in cells expressing the truncated LH/CG receptors. Since hCG-stimulated cAMP stimulation and hCG internalization are retained by rLHR-t631, it can be concluded that the residues, not necessarily the same, required for these functions reside within the 26 amino acids of the cytoplasmic tail closest to the seventh transmembrane helix and/or residues within the intracellular loops. Our data show, however, that both hCG-stimulated cAMP production and hCG internalization are enhanced by the removal of the distal portion of the cytoplasmic tail.     

Identification of the intracellular region of the leukotriene B4 receptor type 1 that is specifically involved in Gi activation

Many G-protein-coupled receptors can activate more than one G-protein subfamily member. Leukotriene B4 receptor type 1 (BLT1) is a high affinity G-protein-coupled receptors for leukotriene B4 functioning in host defense, inflammation, and immunity. Previous studies have shown that BLT1 utilizes different G-proteins (the Gi family and G16 G-proteins) in mediating diverse cellular events and that truncation of the cytoplasmic tail of BLT1 does not impair activation of Gi and G16 proteins. To determine responsive regions of BLT1 for G-protein coupling, we performed an extensive mutagenesis study of its intracellular loops. Three intracellular loops (i1, i2, and i3) of BLT1 were found to be important for both Gi and G16 coupling, as judged by Gi-dependent guanosine 5'-(gamma-thio) triphosphate (GTPgammaS) binding and G16-dependent inositol phosphate accumulation assays. The i3-1 mutant, with a mutation at the i3 amino terminus, exhibited greatly reduced GTPgammaS binding but intact inositol phosphate accumulation triggered by leukotriene B4 stimulation. These results suggest that the i3-1 region is required only for Gi activation. Moreover, in the i3-1 mutant, the deficiency in Gi activation was accompanied by a loss of the high affinity leukotriene B4 binding state seen with the wild type receptor. A three-dimensional model of BLT1 constructed based on the structure of bovine rhodopsin suggests that the i3-1 region may consist of the cytoplasmic end of the transmembrane helix V, which protrudes the helix into the cytoplasm. From mutational studies and three-dimensional modeling, we propose that the extended cytoplasmic helix connected to the transmembrane helix V of BLT1 might be a key region for selective activation of Gi proteins.     

Insights into the structure and molecular basis of ligand docking to the G protein-coupled secretin receptor using charge-modified amino-terminal agonist probes

The amino terminus and third loop regions of class B G protein-coupled receptors play critical roles in ligand docking and action. For the prototypic secretin receptor, the hormone amino terminus is spatially approximated with receptor region high in transmembrane segment 6 (TM6), whereas residues ranging from position 6 through 26 label the amino terminus. Here, we focus on the role of charge of the secretin amino terminus, using a series of full-agonist, acetylated probes. Sites of covalent labeling were examined using sequential purification, chemical and enzymatic cleavage, and Edman degradation. High-affinity amino-terminally-blocked probes labeled the distal amino-terminal tail, rather than TM6, while adding a basic residue, again labeled TM6. These data suggest that the secretin amino terminus docks between the amino terminus and TM6 of the receptor, with this region of secretin likely interacting with an acidic residue within the receptor TM6 and the third extracellular loop. To explore this, candidate acidic residues were mutated to Ala (E341A, D342A, E345A, E351A). The E351A mutant markedly interfered with binding, biological activity, and internalization, whereas all others bound secretin and signaled and internalized normally. This supports the possibility that there is a charge-charge interaction between this residue and the amino terminus of secretin that is critical to its normal docking.     

Disruption of the mu-delta opioid receptor heteromer

The crystal structure of the mu and kappa opioid receptors has revealed dimeric structural arrangements. Mu-delta receptors heteromers also exist and we have identified discrete cytoplasmic regions in each receptor required for oligomer formation. In the carboxyl tail of the delta receptor we identified three glycine residues (-GGG), substitution of any of these residues prevented heteromer formation. In intracellular loop 3 of both mu and delta receptors we identified three residues (-SVR), substitution of any of these residues prevented heteromer formation.     

Molecular characterization of a functional cDNA for rat substance P receptor

This paper describes the amino acid sequence of the rat substance P receptor and its comparison with that of the rat substance K receptor on the basis of molecular cloning and sequence analysis. From a rat brain cDNA library constructed with an RNA expression vector, we identified a cDNA mixture containing a functional substance P receptor cDNA by examining electrophysiologically a receptor expression following injection of the mRNAs synthesized in vitro into Xenopus oocytes. A receptor cDNA clone was then isolated by cross-hybridization with the bovine substance K receptor cDNA. The clone was confirmed by selective binding of substance P to the cloned receptor expressed in mammalian COS cells. The deduced amino acid sequence (407 amino acid residues) possesses seven putative membrane spanning domains and shows a sequence similarity to the members of G-protein-coupled receptors. The rat substance P and substance K receptors are very similar in both size and amino acid sequences, particularly in the putative transmembrane regions and the first and second cytoplasmic loops. This similarity is in marked contrast to the sequence divergence in the amino- and carboxyl-terminal regions and the third cytoplasmic loop. The observed sequence similarity and divergence would thus contribute to the expression of similar but pharmacologically distinguishable activities of the two tachykinin receptors.     

Structures of the intradiskal loops and amino terminus of the G-protein receptor, rhodopsin.

The intradiskal surface of the transmembrane protein, rhodopsin, consists of the amino terminal domain and three loops connecting six of the seven transmembrane helices. This surface corresponds to the extracellular surface of other G-protein receptors. Peptides that represent each of the extramembraneous domains on this surface (three loops and the amino terminus) were synthesized. These peptides also included residues which, based on a hydrophobic plot, could be expected to be part of the transmembrane helix. The structure of each of these peptides in solution was then determined using two-dimensional 1H nuclear magnetic resonance. All peptide domains showed ordered structures in solution. The structures of each of the peptides from intradiskal loops of rhodopsin exhibited a turn in the central region of the peptide. The ends of the peptides show an unwinding of the transmembrane helices to form this turn. The amino terminal domain peptide exhibited alpha-helical regions with breaks and bends at proline residues. This region forms a compact domain. Together, the structures for the loop and amino terminus domains indicate that the intradiskal surface of rhodopsin is ordered. These data further suggest a structural motif for short loops in transmembrane proteins. The ordered structures of these loops, in the absence of the transmembrane helices, indicate that the primary sequences of these loops are sufficient to code for the turn.     

Three distinct epitopes on the extracellular face of the glucagon receptor determine specificity for the glucagon amino terminus

The glucagon and glucagon-like peptide-1 (GLP-1) receptors are homologous family B seven-transmembrane (7TM) G protein-coupled receptors, and they selectively recognize the homologous peptide hormones glucagon (29 amino acids) and GLP-1 (30-31 amino acids), respectively. The amino-terminal extracellular domain of the glucagon and GLP-1 receptors (140-150 amino acids) determines specificity for the carboxyl terminus of glucagon and GLP-1, respectively. In addition, the glucagon receptor core domain (7TM helices and connecting loops) strongly determines specificity for the glucagon amino terminus. Only 4 of 15 residues are divergent in the glucagon and GLP-1 amino termini; Ser2, Gln3, Tyr10, and Lys12 in glucagon and the corresponding Ala8, Glu9, Val16, and Ser18 in GLP-1. In this study, individual substitution of these four residues of glucagon with the corresponding residues of GLP-1 decreased the affinity and potency at the glucagon receptor relative to glucagon. Substitution of distinct segments of the glucagon receptor core domain with the corresponding segments of the GLP-1 receptor rescued the affinity and potency of specific glucagon analogs. Site-directed mutagenesis identified the Asp385 --> Glu glucagon receptor mutant that specifically rescued Ala2-glucagon. The results show that three distinct epitopes of the glucagon receptor core domain determine specificity for the N terminus of glucagon. We suggest a glucagon receptor binding model in which the extracellular ends of TM2 and TM7 are close to and determine specificity for Gln3 and Ser2 of glucagon, respectively. Furthermore, the second extracellular loop and/or proximal segments of TM4 and/or TM5 are close to and determine specificity for Lys12 of glucagon.