Identification of Two Pairs of Spatially Approximated Residues within the Carboxyl Terminus of Secretin and Its Receptor

Abstract

The carboxyl-terminal domains of secretin family peptides have been shown to contain key determinants for high affinity binding to their receptors. In this work, we have examined the interaction between carboxyl-terminal residues within secretin and the prototypic secretin receptor. We previously utilized photoaffinity labeling to demonstrate spatial approximation between secretin residue 22 and the receptor domain that includes the first 30 residues of the amino terminus (Dong, Detailed understanding of the molecular basis of ligand binding and receptor activation will be the key to facilitate the rational design of new drugs. Guanine nucleotide-binding protein (G protein)-coupled receptors represent the largest group of plasma membrane receptors and include many potentially important targets for therapeutic agents, making this superfamily an ideal focus for these efforts.MThis superfamily includes receptor families that share sequence homologies and structural similarities of their natural ligands. Among these is the recently described class II family that includes receptors for secretin, calcitonin, parathyroid hormone, glucagon, vasoactive intestinal polypeptide, and pituitary adenylate cyclase-activating polypeptide (3, 4). The natural agonist ligands of these receptors have helical domains in both carboxyl-terminal and amino-terminal regions (5, 6) and have binding determinants that are spread throughout their entire length (3, 4, 7). The theme is emerging that aminoterminal regions of these peptides contain key determinants for receptor selectivity, whereas carboxyl-terminal regions contain determinants for high affinity binding (8, 9). Of note, the carboxyl-terminal regions of some of the members of this family may even be interchanged while maintaining high affinity binding (9). This feature supports the likely general relevance of the observations in the current work to the entire class II family.We recently used affinity labeling to demonstrate spatial approximation between a photolabile residue within the carboxyl-terminal half of secretin (position 22) and a 30-residue segment at the distal amino terminus of its prototypic secretin receptor (1). Of interest, the amino-terminal tail of the class II G protein-coupled receptor family has remarkable structural features and major functional importance (3,4). This receptor domain is moderately large, containing greater than 110 residues, with six conserved Cys residues and key disulfide bonds (10). Chemical reduction and treatment with sulfhydryl-reactive compounds have had substantial negative impact on these receptors (10 -12). Truncation, site-directed mutagenesis, and chimeric receptor studies have also supported the critical role of this domain in the function of this receptor family (9,13).In the present work, we have extended our understanding of the siting of secretin residue 22 when bound to its receptor by defining a receptor residue adjacent to it. We have also established spatial approximation between another residue closer to the carboxyl ter...

Molecular Approximation between a Residue in the Amino-terminal Region of Calcitonin and the Third Extracellular Loop of the Class B G Protein-coupled Calcitonin Receptor

“…Active Site Identification-To gain an initial indication of receptor domains of labeling with the Bpa 8 probe, we first chose to cleave the labeled receptor by CNBr that we have used successfully for the secretin (22,26,(35)(36)(37)(38)(39) and CT (20) receptors in our laboratory. CNBr cleaves at the carboxyl side of Met residues within a protein, and its cleavage of the CT receptor would theoretically yield 15 fragments ranging from 0.1 to 11 kDa, two of which contain potential glycosylation sites.…”

Secretin Receptor Oligomers Form Intracellularly during Maturation through Receptor Core Domains

“…BFA, formaldehyde and coelenterazine h were purchased from Molecular Probes (Eugene, OR), Ted Pella (Redding, CA) and Biotium (Hayward, CA), respectively. The natural rat secretin and [Tyr 10 ]secretin peptides used in binding assays were synthesized in our laboratory (33) and have been shown to be functional at human secretin receptors in intact cells (34). Oxidative radioiodination of the Tyr 10 residue to form the 125 I-[Tyr 10 ]secretin radioligand has been described elsewhere (33).…”

Agonist-dependent Dissociation of Oligomeric Complexes of G Protein-coupled Cholecystokinin Receptors Demonstrated in Living Cells Using Bioluminescence Resonance Energy Transfer

“…cDNA Constructs for Expression Studies-The rat type A CCK receptor and human secretin receptor cDNA constructs in pcDNA3 were prepared as we have described (17,18). CCK receptor constructs having an epitope tag (influenza hemagglutinin (HA) or FLAG) at its amino terminus were prepared using the QuikChange TM Site-Directed Mutagenesis Kit (Stratagene).…”