Relationship Between Native and Recombinant Cholecystokinin Receptors

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The calcitonin receptor is a member of the class B family of G protein-coupled receptors, which contains numerous potentially important drug targets. Delineation of themes for agonist binding and activation of these receptors will facilitate the rational design of receptor-active drugs. We reported previously that a photolabile residue within the carboxyl-terminal half (resi- Calcitonin (CT), 1 secreted by the thyroid gland in response to elevations in blood calcium levels, is a peptide hormone that regulates calcium by inhibition of osteoclast-mediated bone resorption (1, 2). CT acts on bone and kidney to maintain calcium homeostasis and is also present in the central nervous system, where it has anorectic and analgesic effects (3). It has been used therapeutically for the treatment of Paget's disease, the hypercalcemia associated with certain types of tumors, and osteoporosis (1, 2).dueCT is a relatively large peptide that contains 32 amino acids and has a diffuse pharmacophoric domain. Although residues throughout the entire length have been demonstrated to be critical for its biological activity, the amino-terminal residues of CT contain key determinants for its receptor agonist selectivity (1, 2). Truncation of the first seven amino-terminal residues that includes a disulfide bond between residues 1 and 7 results in antagonist action (4, 5). Residues 8 through 22 tend to form an amphiphilic ␣-helical structure that is important for high affinity binding (1).CT exhibits its agonist activities through binding to the CT receptor, a member of the class B family of guanine nucleotidebinding protein (G protein)-coupled receptors that have the seven-transmembrane-domain structure. Although they have topology similar to that of class A receptors, members of class B family share less than 12% amino acid identity with the more extensively studied receptors in the class A family. Class B receptors have distinct signature sequences, including a long complex amino-terminal domain with six conserved cysteine residues that are believed to be involved in intradomain disulfide bonds critical for establishing functional receptor conformation (6 -10). Members included in this family are receptors for moderately large peptides having diffuse pharmacophoric domains, such as secretin, calcitonin, glucagon, vasoactive intestinal polypeptide, pituitary adenylate cyclase-activating polypeptide, and parathyroid hormone, sharing 30 to 50% homology with each other.The unique amino-terminal domain of the CT receptor has been shown to be critical for agonist binding and receptor activation using chimeric receptor studies (11)(12)(13). This represents a consistent theme for other class B family members (14 -19). Photoaffinity labeling is a more direct approach for exploration of spatial approximations between residues within a ligand and within its receptor. Using this approach, we have recently demonstrated that probes incorporated a photolabile p-benzoyl-L-phenylalanine (Bpa) residue in the carboxyl-terminal half and mid-region of the ...

“…Cells were harvested mechanically 72 h after transfection. Plasma membranes were prepared from the above receptor-expressing cells using methods reported previously (29).…”

Section: Methodsmentioning

confidence: 99%

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

Dong

Pinon

Cox

et al. 2004

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“…These cells were harvested mechanically 72 h after transfection. Receptor-enriched plasma membranes were prepared from the above stable HEK293 cell line and transiently transfected COS cells using methods that we previously reported (23).…”

Section: Methodsmentioning

confidence: 99%

“…After electrophoresis, labeled bands were identified by exposure to x-ray film with intensifying screens at Ϫ80°C. Aliquots of affinity-labeled receptor and relevant receptor fragments were deglycosylated with endoglycosidase F, as described previously (23).…”

Section: Methodsmentioning

confidence: 99%

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

Dong¹,

Pinon²,

Cox

et al. 2004

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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...

“…Ligand Binding Studies-Cell membranes were prepared from secretin receptor-bearing cells using methods we previously reported (20). For binding characterization, membranes (5-10 g) were incubated with a constant amount of radioligand, 125 I-[Tyr 10 ]rat secretin-27 (3-5 pM), in the presence of increasing concentrations of non-radiolabeled secretin analogue (0 -1 M) for 1 h at room temperature in KrebsRingers/HEPES medium (25 mM HEPES, pH 7.4, 104 mM NaCl, 5 mM KCl, 1.2 mM MgSO 4 , 2 mM CaCl 2 , 1 mM KH 2 PO 4 , 0.2% bovine serum albumin, 0.01% soybean trypsin inhibitor, and 1 mM phenylmethylsulfonyl fluoride).…”

Section: Methodsmentioning

confidence: 99%

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

Dong

Asmann

Zang

et al. 2000

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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...