Insights into the impact of phenolic residue incorporation at each position along secretin for receptor binding and biological activity.

Understanding of the structural importance of each position along a peptide ligand can provide important insights into the molecular basis for its receptor binding and biological activity. This has typically been evaluated using serial replacement of each natural residue with an alanine. In the current report, we have further complemented alanine scanning data with the serial replacement of each residue within secretin-27, the natural ligand for the prototypic class B G protein-coupled secretin receptor, using a photolabile phenolic residue. This not only provided the opportunity to probe spatial approximations between positions along a docked ligand with its receptor, but also provided structure-activity insights when compared with tolerance for alanine replacement of the same residues. The pattern of sensitivity to phenolic residue replacement was periodic within the carboxyl-terminal region of this peptide ligand, corresponding with alanine replacements in that region. This was supportive of the alpha-helical conformation of the peptide in that region and its docking within a groove in the receptor amino-terminal domain. In contrast, the pattern of sensitivity to phenolic residue replacement was almost continuous in the amino-terminal region of this peptide ligand, again similar to alanine replacements, however, there were key positions in which either the phenolic residue or alanine was differentially preferred. This provided insights into the receptor environment of the portion of this ligand most critical for its biological activity. As the structure of the intact receptor is elucidated, these data will provide a guide for ligand docking to the core domain and to help clarify the molecular basis of receptor activation.

Glucagon-like peptide-1 receptor dimerization differentially regulates agonist signaling but does not affect small molecule allostery.

The glucagon-like peptide-1 receptor (GLP-1R) is a family B G protein-coupled receptor and an important drug target for the treatment of type II diabetes, with activation of pancreatic GLP-1Rs eliciting glucose-dependent insulin secretion. Currently, approved therapeutics acting at this receptor are peptide based, and there is substantial interest in small molecule modulators for the GLP-1R. Using a variety of resonance energy transfer techniques, we demonstrate that the GLP-1R forms homodimers and that transmembrane helix 4 (TM4) provides the primary dimerization interface. We show that disruption of dimerization using a TM4 peptide, a minigene construct encoding TM4, or by mutation of TM4, eliminates G protein-dependent high-affinity binding to GLP-1(7-36)NH(2) but has selective effects on receptor signaling. There was <10-fold decrease in potency in cAMP accumulation or ERK1/2 phosphorylation assays but marked loss of intracellular calcium mobilization by peptide agonists. In contrast, there was near-complete abrogation of the cAMP response to an allosteric agonist, compound 2, but preservation of ERK phosphorylation. Collectively, this indicates that GLP-1R dimerization is important for control of signal bias. Furthermore, we reveal that two small molecule ligands are unaltered in their ability to allosterically modulate signaling from peptide ligands, demonstrating that these modulators act in cis within a single receptor protomer, and this has important implications for small molecule drug design.

Mapping spatial approximations between the amino terminus of secretin and each of the extracellular loops of its receptor using cysteine trapping.

While it is evident that the carboxyl-terminal region of natural peptide ligands bind to the amino-terminal domain of class B GPCRs, how their biologically critical amino-terminal regions dock to the receptor is unclear. We utilize cysteine trapping to systematically explore spatial approximations among residues in the first five positions of secretin and in every position within the receptor extracellular loops (ECLs). Only Cys(2) and Cys(5) secretin analogues exhibited full activity and retained moderate binding affinity (IC(50): 92±4 and 83±1 nM, respectively). When these peptides probed 61 human secretin receptor cysteine-replacement mutants, a broad network of receptor residues could form disulfide bonds consistent with a dynamic ligand-receptor interface. Two distinct patterns of disulfide bond formation were observed: Cys(2) predominantly labeled residues in the amino terminus of ECL2 and ECL3 (relative labeling intensity: Ser(340), 94±7%; Pro(341), 84±9%; Phe(258), 73±5%; Trp(274) 62±8%), and Cys(5) labeled those in the carboxyl terminus of ECL2 and ECL3 (Gln(348), 100%; Ile(347), 73±12%; Glu(342), 59±10%; Phe(351), 58±11%). These constraints were utilized in molecular modeling, providing improved understanding of the structure of the transmembrane bundle and interconnecting loops, the orientation between receptor domains, and the molecular basis of ligand docking. Key spatial approximations between peptide and receptor predicted by this model (H(1)-W(274), D(3)-N(268), G(4)-F(258)) were supported by mutagenesis and residue-residue complementation studies.

Site of action of a pentapeptide agonist at the glucagon-like peptide-1 receptor. Insight into a small molecule agonist-binding pocket.

The development of small molecule agonists for class B G protein-coupled receptors (GPCRs) has been quite challenging. With proof-of-concept that exenatide, the parenterally administered peptide agonist of the glucagon-like peptide-1 (GLP1) receptor, is an effective treatment for patients with diabetes mellitus, the development of small molecule agonists could have substantial advantages. We previously reported a lead for small molecule GLP1 receptor agonist development representing the pentapeptide NRTFD. In this work, we have prepared an NRTFD derivative incorporating a photolabile benzoylphenylalanine and used it to define its site of action. This peptide probe was a full agonist with potency similar to NRTFD, which bound specifically and saturably to a single, distinct site within the GLP1 receptor. Peptide mapping using cyanogen bromide and endoproteinase Lys-C cleavage of labeled wild type and M397L mutant receptor constructs identified the site of covalent attachment of NRTFD within the third extracellular loop above the sixth transmembrane segment (TM6). This region is the same as that identified using an analogous photolabile probe based on secretin receptor sequences, and has been shown in mutagenesis studies to be important for natural agonist action of several members of this family. While these observations suggest that small molecule ligands can act at a site bordering the third extracellular loop to activate this class B GPCR, the relationship of this site to the site of action of the amino-terminal end of the natural agonist peptide is unclear.

Lactam constraints provide insights into the receptor-bound conformation of secretin and stabilize a receptor antagonist.

The natural ligands for family B G protein-coupled receptors are moderate-length linear peptides having diffuse pharmacophores. The amino-terminal regions of these ligands are critical for biological activity, with their amino-terminal truncation leading to production of orthosteric antagonists. The carboxyl-terminal regions of these peptides are thought to occupy a ligand-binding cleft within the disulfide-bonded amino-terminal domains of these receptors, with the peptides in amphipathic helical conformations. In this work, we have characterized the binding and activity of a series of 11 truncated and lactam-constrained secretin(5-27) analogues at the prototypic member of this family, the secretin receptor. One peptide in this series with lactam connecting residues 16 and 20 [c[E(16),K(20)][Y(10)]sec(5-27)] improved the binding affinity of its unconstrained parental peptide 22-fold while retaining the absence of endogenous biological activity and competitive antagonist characteristics. Homology modeling with molecular mechanics and molecular dynamics simulations established that this constrained peptide occupies the ligand-binding cleft in an orientation similar to that of natural full-length secretin and provided insights into why this peptide was more effective than other truncated conformationally constrained peptides in the series. This lactam bridge is believed to stabilize an extended α-helical conformation of this peptide while in solution and not to interfere with critical residue-residue approximations while docked to the receptor.

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.

Refinement of glucagon-like peptide 1 docking to its intact receptor using mid-region photolabile probes and molecular modeling.

The glucagon-like peptide 1 (GLP1) receptor is an important drug target within the B family of G protein-coupled receptors. Its natural agonist ligand, GLP1, has incretin-like actions and the receptor is a recognized target for management of type 2 diabetes mellitus. Despite recent solution of the structure of the amino terminus of the GLP1 receptor and several close family members, the molecular basis for GLP1 binding to and activation of the intact receptor remains unclear. We previously demonstrated molecular approximations between amino- and carboxyl-terminal residues of GLP1 and its receptor. In this work, we study spatial approximations with the mid-region of this peptide to gain insights into the orientation of the intact receptor and the ligand-receptor complex. We have prepared two new photolabile probes incorporating a p-benzoyl-l-phenylalanine into positions 16 and 20 of GLP1(7-36). Both probes bound to the GLP1 receptor specifically and with high affinity. These were each fully efficacious agonists, stimulating cAMP accumulation in receptor-bearing CHO cells in a concentration-dependent manner. Each probe specifically labeled a single receptor site. Protease cleavage and radiochemical sequencing identified receptor residue Leu(141) above transmembrane segment one as its site of labeling for the position 16 probe, whereas the position 20 probe labeled receptor residue Trp(297) within the second extracellular loop. Establishing ligand residue approximation with this loop region is unique among family members and may help to orient the receptor amino-terminal domain relative to its helical bundle region.

Importance of each residue within secretin for receptor binding and biological activity.

Secretin is a linear 27-residue peptide hormone that stimulates pancreatic and biliary ductular bicarbonate and water secretion by acting at its family B G protein-coupled receptor. While, like other family members, the carboxyl-terminal region of secretin is most important for high affinity binding and its amino-terminal region is most important for receptor selectivity and receptor activation, determinants for these activities are distributed throughout the entire length of this peptide. In this work, we have systematically investigated changing each residue within secretin to alanine and evaluating the impact on receptor binding and biological activity. The residues most critical for receptor binding were His1, Asp3, Gly4, Phe6, Thr7, Ser8, Leu10, Asp15, Leu19, and Leu23. The residues most critical for biological activity included His1, Gly4, Thr7, Ser8, Glu9, Leu10, Leu19, Leu22, and Leu23, with Asp3, Phe6, Ser11, Leu13, Asp15, Leu26, and Val27 also contributing. While the importance of residues in positions analogous to His1, Asp3, Phe6, Thr7, and Leu23 is conserved for several closely related members of this family, Leu19 is uniquely important for secretin. We, therefore, have further studied this residue by molecular modeling and molecular dynamics simulations. Indeed, the molecular dynamics simulations showed that mutation of Leu19 to alanine was destabilizing, with this effect greater than that observed for the analogous position in the other close family members. This could reflect reduced contact with the receptor or an increase in the solvent-accessible surface area of the hydrophobic residues in the carboxyl terminus of secretin as bound to its receptor.

Elucidation of the active conformation of the amino terminus of receptor-bound secretin using intramolecular disulfide bond constraints.

Family B G protein-coupled receptors include several potentially important drug targets, yet our understanding of the molecular basis of ligand binding to and activation of these receptors is incomplete. While NMR and crystal structures exist for peptide ligand-associated amino-terminal domains of several family members, these only provide insights into the conformation of the carboxyl-terminal region of the peptides. The amino-terminal region of these peptides, critical for biological activity, is believed to interact with the helical bundle domain, and is, therefore, unconstrained in these structures. The aim of the current study was to provide insights into the conformation of the amino terminus of secretin as bound to its receptor. We prepared a series of conformationally constrained secretin peptides containing intramolecular disulfide bonds that were predicted by molecular modeling to approximate the conformation of the analogous region of PACAP bound to its receptor that had been determined using transfer-NOE NMR techniques. Secretin peptides with pairs of cysteine residues in positions 2-7, 3-5, 3-6, 4-7, 7-9, and 4-10 were studied as linear and disulfide-bonded forms. The analog with a disulfide bond connecting positions 7-9 had binding affinity and biological activity similar to natural secretin, supporting the relevance of this constraint to its active conformation. While this feature is shared between secretin and PACAP, absence of activity in other constrained peptides in this series also suggest that there are differences between these receptor-bound conformations. It will be critical to extend similar studies to other family members to learn what structural elements might be most conserved in this family.

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 Tyr(145), adjacent to the first transmembrane segment, as the site of labeling by the position 12 probe, and receptor residue Tyr(205), 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.

Refinement of the pharmacophore of an agonist ligand of the secretin receptor using conformationally constrained cyclic hexapeptides.

There is a compelling need for the development of small molecule agonists acting at family B G protein-coupled receptors. A possible lead for the development of such drugs was reported when it was recognized that sequences endogenous to the amino terminus of the secretin receptor and certain other receptors in this family possess weak full agonist activity (Dong et al. Mol Pharmacol 2006;70:206-213). In the current report, we extended those observations by building the active dipeptide motif found in the secretin receptor (WD) into each position around a conformationally constrained d-amino acid-containing cyclic hexapeptide, and determining the biological activity of each peptide at the secretin receptor. Indeed, only two positions for WD around this constrained ring resulted in biological activity at the receptor, providing further insights into the structural specificity of this phenomenon. Molecular modeling supported the presence of a unique WD backbone conformation shared only by these active peptides, and provided a more constrained template for future receptor-active agonist drug development.

Secretin occupies a single protomer of the homodimeric secretin receptor complex: insights from photoaffinity labeling studies using dual sites of covalent attachment.

The secretin receptor, a prototypic family B G protein-coupled receptor, forms a constitutive homodimeric complex that is stable even in the presence of hormone. Recently, a model of this agonist-bound receptor was built based on high resolution structures reported for amino-terminal domains of other family members. Although this model provided the best solution for all extant data, including 10 photoaffinity labeling constraints, a new such constraint now obtained with a position 16 photolabile probe was inconsistent with this model. As the secretin receptor forms constitutive homodimers, we explored whether secretin might dock across both protomers of the complex, an observation that could also contribute to the negative cooperativity observed. To directly explore this, we prepared six secretin analogue probes that simultaneously incorporated two photolabile benzoylphenylalanines as sites of covalent attachment, in positions known to label distinct receptor subdomains. Each bifunctional probe was a full agonist that labeled the receptor specifically and saturably, with electrophoretic migration consistent with labeling a single protomer of the homodimeric secretin receptor. No band representing radiolabeled receptor dimer was observed with any bifunctional probe. The labeled monomeric receptor bands were cleaved with cyanogen bromide to demonstrate that both of the photolabile benzoylphenylalanines within a single probe had established covalent adducts with a single receptor in the complex. These data are consistent with a model of secretin occupying a single secretin receptor protomer within the homodimeric receptor complex. A new molecular model accommodating all constraints is now proposed.

Molecular basis of glucagon-like peptide 1 docking to its intact receptor studied with carboxyl-terminal photolabile probes.

The glucagon-like peptide 1 (GLP1) receptor is a member of Family B G protein-coupled receptors and represents an important drug target for type 2 diabetes. Despite recent solution of the structure of the amino-terminal domain of this receptor and that of several close family members, understanding of the molecular basis of natural ligand GLP1 binding to its intact receptor remains limited. The goal of this study was to explore spatial approximations between specific receptor residues within the carboxyl terminus of GLP1 and its receptor as normally docked. Therefore, we developed and characterized two high affinity, full-agonist photolabile GLP1 probes having sites for covalent attachment in positions 24 and 35. Both probes labeled the receptor specifically and saturably. Subsequent peptide mapping using chemical and proteinase cleavages of purified wild-type and mutant GLP1 receptor identified that the Arg(131)-Lys(136) segment at the juxtamembrane region of the receptor amino terminus contained the site of labeling for the position 24 probe, and the specific receptor residue labeled by this probe was identified as Glu(133) by radiochemical sequencing. Similarly, nearby residue Glu(125) within the same region of the receptor amino-terminal domain was identified as the site of labeling by the position 35 probe. These data represent the first direct demonstration of spatial approximation between GLP1 and its intact receptor as docked, providing two important constraints for the modeling of this interaction. This should expand our understanding of the molecular basis of natural agonist ligand binding to the GLP1 receptor and may be relevant to other family members.

Elucidation of the molecular basis of cholecystokinin Peptide docking to its receptor using site-specific intrinsic photoaffinity labeling and molecular modeling.

G protein-coupled receptors represent the largest family of receptors and the major target of current drug development efforts. Understanding of the mechanisms of ligand binding and activation of these receptors remains limited, despite recent advances in structural determination of family members. This work focuses on the use of photoaffinity labeling and molecular modeling to elucidate the structural basis of binding a natural peptide ligand to a family A G protein-coupled receptor, the type 1 cholecystokinin receptor. Two photolabile cholecystokinin analogues were developed and characterized as representing high-affinity, fully biologically active probes with sites of covalent attachment at positions 28 and 31. The sites of receptor labeling were identified by purification, proteolytic peptide mapping, and radiochemical sequencing of labeled wild-type and mutant cholecystokinin receptors. The position 28 probe labeled second extracellular loop residue Leu(199), while the position 31 probe labeled first extracellular loop residue Phe(107). Along with five additional spatial approximation constraints coming from previous photoaffinity labeling studies and 12 distance restraints from fluorescence resonance energy transfer studies, these were built into two homology models of the cholecystokinin receptor, based on the recent crystal structures of the beta2-adrenergic receptor and A2a-adenosine receptor. The resultant agonist ligand-occupied receptor models fully accommodate all existing experimental data and represent the best refined models of a peptide hormone receptor in this important family.

Use of multidimensional fluorescence resonance energy transfer to establish the orientation of cholecystokinin docked at the type A cholecystokinin receptor.

Fluorescence resonance energy transfer (FRET) represents a powerful tool to establish relative distances between donor and acceptor fluorophores. By utilizing several donors situated in distinct positions within a docked full agonist ligand and several acceptors distributed at distinct sites within its receptor, multiple interdependent dimensions can be determined. These can provide a unique method to establish or confirm three-dimensional structure of the molecular complex. In this work, we have utilized full agonist analogues of cholecystokinin (CCK) with Aladan distributed throughout the pharmacophore in positions 24, 29, and 33, along with receptor constructs derivatized with Alexa (546) at positions 94, 102, 204, and 341 in the helical bundle and first, second, and third extracellular loops, respectively. These provided 12 FRET distances to overlay on working models of the CCK-occupied receptor. These established that the carboxyl terminus of CCK resides at the external surface of the lipid bilayer, adjacent to the receptor amino-terminal tail, rather than being inserted into the helical bundle. They also provide important experimentally derived constraints for understanding spatial relationships between the docked ligand and the flexible extracellular loop regions. Multidimensional FRET provides a new independent method to establish and refine structural insights into ligand-receptor complexes.

Spatial approximation between secretin residue five and the third extracellular loop of its receptor provides new insight into the molecular basis of natural agonist binding.

The amino terminus of class II G protein-coupled receptors plays an important role in ligand binding and receptor activation. Understanding of the conformation of the amino-terminal domain of these receptors has been substantially advanced with the solution of nuclear magnetic resonance and crystal structures of this region of receptors for corticotrophin-releasing factor, pituitary adenylate cyclase-activating polypeptide, and gastric inhibitory polypeptide. However, the orientation of the amino terminus relative to the receptor core and how the receptor gets activated upon ligand binding remain unclear. In this work, we have used photoaffinity labeling to identify a critical spatial approximation between residue five of secretin and a residue within the proposed third extracellular loop of the secretin receptor. This was achieved by purification, deglycosylation, cyanogen bromide cleavage, and sequencing of labeled wild-type and mutant secretin receptors. This constraint has been used to refine our evolving molecular model of secretin docked at the intact receptor, which for the first time includes refined helical bundle and loop regions and reflects a peptide-binding groove within the receptor amino terminus that directs the amino terminus of the peptide toward the receptor body. This model is fully consistent with the endogenous agonist mechanism for class II G protein-coupled receptor activation, where ligand binding promotes the interaction of a portion of the receptor amino terminus with the receptor body to activate it.

Exploration of the endogenous agonist mechanism for activation of secretin and VPAC1 receptors using synthetic glycosylated peptides.

Current understanding of the molecular basis of activation of class II G protein-coupled receptors remains limited, despite recent solution of NMR and crystal structures of amino-terminal domains of several family members. One mechanism proposed for the activation of these receptors involves an agonist-stimulated change in conformation of the receptor amino terminus. This results in the exposure of a "hidden endogenous agonist" (WDN sequence in secretin and VPAC1 receptors) within the receptor amino terminus that interacts with the receptor core, thereby changing its conformation and exposing its G protein-binding region. The Asn in this WDN sequence is known to be glycosylated in both secretin and VPAC1 receptors, raising concern about whether this posttranslational modification might interfere with the proposed mechanism. Therefore, we prepared glycosylated forms of cyclic WDN and the longer cyclic peptide, LWDNM, and tested them for agonist activity at secretin and VPAC1 receptor-bearing cell lines. Both glycosylated peptides stimulated full cAMP responses in the cell lines. Clearly, glycosylation did not interfere with this mechanism and may actually facilitate the correct orientation of the pharmacophore of the endogenous agonist ligand. These data provide further evidence for this proposed mechanism for the activation of this family of receptors.

Insights into the structural basis of endogenous agonist activation of family B G protein-coupled receptors.

Agonist drugs targeting the glucagon-like peptide-1 (GLP1) receptor represent important additions to the clinical management of patients with diabetes mellitus. In the current report, we have explored whether the recently described concept of a receptor-active endogenous agonist sequence within the amino terminus of the secretin receptor may also be applicable to the GLP1 receptor. If so, this could provide a lead for the development of additional small molecule agonists targeting this and other important family members. Indeed, the region of the GLP1 receptor analogous to that containing the active WDN within the secretin receptor was found to possess full agonist activity at the GLP1 receptor. The minimal fragment within this region that had full agonist activity was NRTFD. Despite having no primary sequence identity with the WDN, it was also active at the secretin receptor, where it had similar potency and efficacy to WDN, suggesting common structural features. Molecular modeling demonstrated that an intradomain salt bridge between the side chains of arginine and aspartate could yield similarities in structure with cyclic WDN. This directly supports the relevance of the endogenous agonist concept to the GLP1 receptor and provides new insights into the rational development and refinement of new types of drugs activating this important receptor.

Transmembrane segment IV contributes a functionally important interface for oligomerization of the Class II G protein-coupled secretin receptor.

Oligomerization of the Class II G protein-coupled secretin receptor has been reported, but the molecular basis for this and its functional significance have not been determined. In the current work, we have examined the possible contribution of each of the transmembrane (TM) segments of this receptor to its homo-oligomerization, using the method of competitive disruption screening for inhibition of receptor bioluminescence resonance energy transfer signal. TM IV was the only segment that was found to disrupt receptor bioluminescence resonance energy transfer. Evaluation of predicted interhelical and lipid-exposed faces of this TM segment demonstrated that its lipid-exposed face represented the determinant for oligomerization. This was further confirmed by mutagenesis of the intact secretin receptor. Morphological FRET was utilized to demonstrate that secretin receptor oligomerization occurred at the cell surface and that this oligomerization was disrupted by mutating Gly(243) and Ile(247), key residues within the lipid-exposed face of TM IV. Although disruption of the receptor oligomerization interface had no effect on secretin binding parameters, it reduced the ability of secretin to stimulate intracellular cAMP. This supports a clear functional effect of oligomerization of this receptor. Such an effect might be particularly relevant to clinical situations in which this receptor is overexpressed, such as in certain neoplasms.

Molecular approximations between residues 21 and 23 of secretin and its receptor: development of a model for peptide docking with the amino terminus of the secretin receptor.

The structurally unique amino-terminal domain of class II G protein-coupled receptors is critically important for ligand binding and receptor activation. Understanding the precise role it plays requires detailed insights into the molecular basis of its ligand interactions and the conformation of the ligand-receptor complex. In this work, we used two high-affinity, full-agonist, secretin-like photolabile probes having sites for covalent attachment in positions 21 and 23 and used sequential proteolysis and sequencing of the labeled region of the receptor to identify two new spatial approximation constraints. The position 21 probe labeled receptor residue Arg(15), whereas the position 23 probe labeled receptor residue Arg(21). A homology model of the amino-terminal domain of the secretin receptor was developed using the NMR structure of the analogous domain of the corticotropin-releasing factor receptor. This was attached to a homology model of the secretin receptor transmembrane bundle, with the two domains oriented relative to each other based on continuity of the peptide backbone and by imposing a distance restraint recently identified between the amino-terminal WDN sequence and the region of the helical bundle above transmembrane segment six. Secretin was docked to this model using seven sets of spatial approximation constraints identified in previous photoaffinity labeling studies. This model was found to fully accommodate all existing constraints, as well as the two new approximations identified in this work.