]secretin-27 probe was a fully efficacious agonist, with a potency to stimulate cAMP accumulation by Chinese hamster ovary SecR cells similar to that of natural secretin (EC 50 ‫؍‬ 68 ؎ 22 pM analogue and 95 ؎ 25 pM secretin). It bound specifically and with high affinity (K i ‫؍‬ 5.0 ؎ 1.1 nM) and covalently labeled the M r ‫؍‬ 57,000-62,000 secretin receptor. Cyanogen bromide cleavage of the receptor yielded a major labeled fragment of apparent M r ‫؍‬ 19,000 that shifted to M r ‫؍‬ 9,000 after deglycosylation. This was most consistent with either of two glycosylated domains within the amino-terminal tail of the receptor. Immunoprecipitation with antibody directed to epitope tags incorporated into each of the candidate domains established that the fragment at the amino terminus of the receptor was the site of labeling. This was further localized to the amino-terminal 30 residues of the receptor by additional proteolysis of this fragment with endoproteinase Lys-C. This provides the first direct demonstration of a contact between a secretin-like agonist and its receptor and will contribute a useful constraint to the modeling of this interaction.The secretin receptor is prototypic of a recently recognized family (Class II) of guanine nucleotide-binding protein (G protein) 1 -coupled receptors (1). Members of this family are believed to have the seven-transmembrane segment topology typical of the superfamily, but they share Ͻ12% homology with the extensively studied Class I receptors in the rhodopsin/␤-adrenergic receptor family, and they lack the signature sequences of this family (2, 3). Secretin family receptors have long aminoterminal domains incorporating six highly conserved Cys residues, believed to contribute to disulfide bonds that help define the family (3, 4). Indeed, this complex domain has been suggested to play a key role in agonist binding, as suggested by receptor mutagenesis studies (5-9). Other extracellular loop domains have also been implicated in complementary roles for agonist binding and receptor activation (4,5,7,10). Natural ligands for this family of receptors are all peptides longer than 27 residues, with structure-activity series suggesting the presence of diffuse pharmacophoric domains (3). Although this large diffuse pharmacophore nicely complements the multiple domains predicted to be outside the membrane bilayer, there is no working model to predict how the two molecules might interact.In this work, we attempt to establish an initial constraint that will contribute to the development of a model for the interaction of secretin with its receptor. We do this through photoaffinity labeling. This has the theoretical advantage of directly probing the domain adjacent to the photolabile residue within the probe after it binds to the receptor. Using this approach, we have successfully identified two binding contacts between photolabile analogues of cholecystokinin and its receptor (11,12).In this work, we have developed an analogue of secretin that incorporates a site for radioiodination an...

Distinct spatial approximations between residues within the secretin pharmacophore and its receptor can provide important constraints for modeling this agonist-receptor complex. We previously used a series of probes incorporating photolabile residues into positions 6, 12, 13, 14, 18, 22, and 26 of the 27-residue peptide and demonstrated that each covalently labeled a site within the receptor amino terminus. Although supporting a critical role of this domain for ligand binding, it does not explain the molecular mechanism of receptor activation. Here, we developed probes having photolabile residues at the amino terminus of secretin to explore possible approximations with a different receptor domain. The first probe incorporated a photolabile pbenzoyl-L-phenylalanine into the position of His 1 of rat secretin ([Bpa 1 ,Tyr 10 ]secretin-27). Because His 1 is critical for function, we also positioned a photolabile Bpa as an amino-terminal extension, in positions ؊1 (rat [Bpa ؊1 ,Tyr 10 ]secretin-27) and ؊2 (rat [Bpa ؊2 ,Gly ؊1 ,-Tyr 10 ]secretin-27). Each analog was shown to be a full agonist, stimulating cAMP accumulation in receptorbearing Chinese hamster ovary-SecR cells in a concentration-dependent manner, with the position ؊2 probe being most potent. They bound specifically and saturably, although the position 1 analog had lowest affinity, and all were able to label the receptor efficiently. Sequential specific cleavage, purification, and sequencing demonstrated that the sites of covalent attachment for each probe were high within the sixth transmembrane segment. This suggests that secretin binding may exert tension between the receptor amino terminus and the transmembrane domain to elicit a conformational change effecting receptor activation.The secretin receptor is prototypic of the Class B family of guanine nucleotide-binding protein (G protein)-coupled receptors which includes many important drug targets. Understanding of the molecular basis of ligand binding of receptors is important for the rational design of receptor-active drugs. However, the molecular basis of ligand binding of Class B receptors is less well understood than that of members of the Class A family, such as rhodopsin and the adrenergic receptor. This likely reflects the facts that natural ligands for Class B G protein-coupled receptors are relatively large peptides with diffuse phamacophoric domains and that these receptors have long and complex amino-terminal domains that are important for binding. Both of these interacting domains are flexible, with active conformations that have not been clearly defined.One of the distinct characteristics of the Class B receptor family is the long amino terminus that exceeds 120 residues in length. It contains 6 conserved Cys residues that have been demonstrated to form intradomain disulfide bonds (1-5) and to be critical for ligand binding. These disulfide bonds could provide key constraints for building a model of the secretin receptor, but definitive mapping of these bonds in an active receptor has not...

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]rat secretin-27) that incorporates a photolabile p-benzoyl-Lphenylalanine (Bpa) residue into position 6 of the amino-terminal half of the ligand and used this to identify a specific receptor residue proximate to it. This probe specifically bound to the secretin receptor with high affinity (IC 50 ‫؍‬ 13.2 ؎ 2.5 nM) and was a potent stimulant of cAMP accumulation in secretin receptor-bearing Chinese hamster ovary-SecR cells (EC 50 ‫؍‬ 720 ؎ 230 pM). It covalently labeled the secretin receptor in a saturable and specific manner. Cyanogen bromide cleavage of this molecule yielded a single labeled fragment that migrated on an SDS-polyacrylamide gel at M r ‫؍‬ 19,000 that shifted to 10 after deglycosylation, most consistent with either of two glycosylated fragments within the aminoterminal tail. By immunoprecipitation with antibody directed to epitope tags incorporated into each of the two candidate fragments, the most distal fragment at the amino terminus was identified as the domain of labeling. The labeled domain was further refined to the first 16 residues by endoproteinase Lys-C cleavage and by cyanogen bromide cleavage of another receptor construct in which Val 16 was mutated to Met. Radiochemical sequencing of photoaffinity-labeled secretin receptor fragments established that Val 4 was the specific site of covalent attachment. This provides the first residueresidue contact between a secretin ligand and its receptor and will contribute substantially to the molecular understanding of this interaction.The secretin receptor is a prototypic member of the Class II family within the superfamily of guanine nucleotide-binding protein (G protein)-coupled receptors (2). These are structurally distinct from the Class I receptors in the rhodopsin/␤-adrenergic receptor family, sharing their heptahelical topology but having distinct signature sequences and a unique extended amino-terminal motif with six conserved Cys residues (3, 4). The Class II receptors all bind moderately large peptides that have diffuse pharmacophoric domains (3, 4). The molecular basis of ligand binding to these receptors is not well understood.At the present time, broad receptor domains of importance in ligand binding have been identified. This is based predominantly on mutagenesis studies, including analysis of chimeric receptors, deletion constructs, and site mutants (5-13). These studies have postulated a critical role for the amino-terminal domain of the Class II receptors in binding their natural peptide ligands. Application of these approaches to the secretin receptor have been consistent with observations with other family members (8 -10). The indirect insights provided by the mutagenesis studies have been supported by the more direct analysis of interacting domains by affinity labeling (1,14,15). Included among these studies is the first demonstration of the affinity labeling of a segment of the amino terminus of the secretin receptor that represents the first 30 residues using a probe with its photolabile site of attachment in position 22 in ...

The class B family of G protein-coupled receptors contains several potentially important drug targets, yet our understanding of the molecular basis of ligand binding and receptor activation remains incomplete. Although a key role is recognized for the cysteine-rich, disulfide-bonded amino-terminal domain of these receptors, detailed insights into ligand docking and resultant conformational changes are not clear. We postulate that binding natural ligands to this domain results in a conformational change that exposes an endogenous ligand which interacts with the body of the receptor to activate it. In this work, we examined whether a synthetic peptide corresponding to a candidate region between the first and third conserved cysteines could act as an agonist. Indeed, this peptide was a weakly potent but fully efficacious agonist, stimulating a concentration-dependent cAMP response in secretin receptorbearing cells. This effect was maintained as the peptide length was reduced from 30 to 5, and ultimately, three residues focused on the conserved residue Asp 49 . The agonist potency was enhanced by cyclization through a diaminopropionic acid linker and by amino-terminal fatty acid acylation. Both ends of the cyclic peptide were shown to interact with the top of transmembrane segment 6 of the receptor, using probes with a photolabile benzoyl-phenylalanine on each end. Analogous observations were also made for two other members of this family, the vasoactive intestinal polypeptide type 1 and calcitonin receptors. These data may provide a unique molecular mechanism and novel leads for the development of smallmolecule agonists acting at potential drug targets within this physiologically important receptor family.G protein-coupled receptors represent the largest group of receptor molecules, with natural ligands ranging from small photons, odorants, and biogenic amines to larger peptides and glycoproteins, and even very large viral particles (Ji et al., 1998). Agonist ligands bind to receptor domains ranging from the intramembranous confluence of helices to the surface loops and amino-terminal domains that vary substantially among distinct groups of receptors within this superfamily (Schwartz and Rosenkilde, 1996). In general, as ligand size increases, the site of its binding moves from the intramembranous domain to the surface and to external domains of its receptor, yet all agonists elicit a conformational change that is reflected in G protein association with the cytosolic face of the receptor. Best understood are the molecular mechanisms for binding the smallest ligands, such as those which occur at rhodopsin (Palczewski et al., 2000).The secretin receptor is prototypic of the class B family of G protein-coupled receptors that includes numerous potentially important drug targets, such as the receptors for parathyroid hormone, calcitonin, glucagon, and vasoactive intestinal polypeptide (VIP) (Ulrich et al., 1998). The natural ligands for these receptors are all moderately large peptides having diffuse pharmacopho...