The C-terminus ends of secretin and VIP interact with the N-terminal domains of their receptors

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...

Section: Discussionmentioning

confidence: 61%

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

Dong

Asmann

Zang

et al. 2000

“…In fact this domain has been shown to be critical for ligand binding by analysis of calcitonin-glucagon (9, 10) and calcitonin-PTH (8) receptor chimeras. The importance of the amino-terminal domain in ligand binding has been consistent for other members in the class B G protein-coupled receptor family, including receptors for secretin (22, 38 -41), vasoactive intestinal polypeptide (VIP) (38,40), PTH (42), and pituitary adenylate cyclase-activating polypeptide (43,44). This is also the domain labeled in analogous photoaffinity labeling studies for mapping of the binding domains of the secretin receptor (11, 12, 14 -17) (for review, see Ref.…”

Section: Identification Of Domains Of Labeling By Peptidementioning

confidence: 92%

Importance of the Amino Terminus in Secretin Family G Protein-coupled Receptors

Dong¹,

Pinon²,

Cox

et al. 2004

The calcitonin receptor is a member of the class B family of G protein-coupled receptors, closely related to secretin and parathyroid hormone receptors. Although mechanisms of ligand binding have been directly explored for those receptors, current knowledge of the molecular basis of calcitonin binding to its receptor is based only on receptor mutagenesis. In this work we have utilized the more direct approach of photoaffinity labeling to explore spatial approximations between distinct residues within calcitonin and its receptor. For this we have developed two human calcitonin analogues incorporating a photolabile p-benzoyl-L-phenylalanine residue in the mid-region and carboxyl-terminal half of the peptide in positions 16 and 26, respectively. Both probes specifically bound to the human calcitonin receptor with high affinity and were potent stimulants of cAMP accumulation in calcitonin receptor-bearing human embryonic kidney 293 cells. They covalently labeled the calcitonin receptor in a saturable and specific manner. Further purification, deglycosylation, specific chemical and enzymatic cleavage, and sequencing of labeled wild type and mutant calcitonin receptors identified the sites of labeling for the position 16 and 26 probes as receptor residues Phe 137 and Thr 30 , respectively. Both were within the extracellular amino terminus of the calcitonin receptor, with the former adjacent to the first transmembrane segment and the latter within the distal amino-terminal tail of the receptor. These data are consistent with affinity labeling of other members of the class B G protein-coupled receptors using analogous probes and may suggest a common ligand binding mechanism for this family.Calcitonin, a hypocalcemic peptide hormone, is secreted from the thyroid gland in response to elevations in serum calcium levels. Its hypocalcemic effect is mediated by inhibition of bone resorption by osteoclasts and enhancement of renal calcium excretion. These actions are important for its widespread clinical use for treatment of bone disorders, including Paget's disease, osteoporosis, and hypercalcemia of malignancy (1, 2).The calcitonin peptide consists of 32 amino acids, with a disulfide bond between residues 1 and 7 which is conserved among all species and that is believed to be critical for its agonist activity. The amino acid sequence and biological potency of calcitonin vary considerably from species to species, but the integrity of the disulfide bond and the carboxyl-terminal proline-amide are necessary for full biological activity (1-3). The disulfide bond can be replaced by other covalent bonds that lead to improved biological stability while retaining full potency (1). The sequence within the amino-terminal loop region is highly conserved in a variety of species but demonstrates divergence in the rest of the sequence (4). Like secretin and peptides for other members of class B G protein-coupled receptor family, the amino-terminal region of calcitonin contains key determinants for receptor agonist selectivity, whereas the...

“…This domain has been suggested to interact with the carboxyl-terminal region of the natural peptide ligands, based on structure-activity relationship, site-directed mutagenesis, and chimeric receptor studies (5)(6)(7)(8)(9)(10), and this is a consistent theme throughout their family.…”

mentioning

confidence: 99%

Molecular Basis of Glucagon-like Peptide 1 Docking to Its Intact Receptor Studied with Carboxyl-terminal Photolabile Probes

Chen

Pinon

Miller

et al. 2009

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.G protein-coupled receptors (GPCRs) 2 are the largest group of membrane receptors with seven transmembrane domains and represent targets for over 30% of approved drugs. Understanding of the molecular basis of ligand binding and activation of these receptors will facilitate the development and refinement of drugs acting at these targets. Currently, the molecular basis of ligand binding and activation of Family B GPCRs is less well understood than that of the larger Family A, where high resolution crystal structures have recently been described for several members (1-4).A characteristic structural feature of this family is a long and structurally complex extracellular amino-terminal domain containing six conserved cysteine residues that form disulfide bonds important for stabilizing the folded structure. This domain has been suggested to interact with the carboxyl-terminal region of the natural peptide ligands, based on structure-activity relationship, site-directed mutagenesis, and chimeric receptor studies (5-10), and this is a consistent theme throughout their family.Insights into the structure of the predominant ligand binding domain, the amino terminus of the Family B GPCRs, have substantially advanced with the solution of NMR and crystal structures of the isolated ligand-bound amino terminus of the receptors for corticotrophin-releasing factor (11-13...