Photoaffinity Cross-linking Identifies Differences in the Interactions of an Agonist and an Antagonist with the Parathyroid Hormone/Parathyroid Hormone-related Protein Receptor

Behar, Vered; Bisello, Alessandro; Bitan, Gal; Rosenblatt, Michael; Chorev, Michael

doi:10.1074/jbc.275.1.9

Cited by 106 publications

(157 citation statements)

References 49 publications

(33 reference statements)

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“…receptors for follicle-stimulating hormone (56,57), luteinizing hormone/choriogonadotropin (52), and gonadotropin-releasing hormone (58)) to activate these receptors. It is noteworthy that the third extracellular loop of G protein-coupled receptors is near the sixth transmembrane domain, which has been shown to interact directly with the amino terminus of the peptide ligand for receptors for parathyroid hormone (23,24) and secretin (22).…”

Section: Resultsmentioning

confidence: 99%

“…This new pair of residue-residue approximation should contribute substantially to our understanding of the natural ligand-binding region of the CT receptor. Analogous observations with the structurally related secretin (22) and parathyroid hormone (23,24) receptors may suggest a common mechanism for ligand binding and activation of the class B family of G protein-coupled receptors.…”

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confidence: 98%

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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

Journal of Biological Chemistry

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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 ...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 98%

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

Journal of Biological Chemistry

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“…It is also similar to covalent labeling of the distal amino-terminal tail of the PTH 1 receptor by PTH/PTHrP probes, incorporating a photolabile residue in positions 23 (47) and 28 (46) (Table I). The identification of the amino-terminal domain of the calcitonin receptor as the labeling domain for both Bpa 16 and Bpa 26 probes is distinct from photoaffinity labeling of the first extracellular loop domain of the PTH 1 receptor by a position 27 probe (51) and from that of the top of the sixth transmembrane domains of the PTH 1 receptor by PTH/PTHrP probes incorporating a photolabile residue at their amino termini (52,53) (Table I).…”

Section: Identification Of Domains Of Labeling By Peptidementioning

confidence: 99%

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

Dong¹,

Pinon²,

Cox

et al. 2004

Journal of Biological Chemistry

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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...

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“…The Ser1 residue used in the simulations adopts a position equidistant between the extracellular portion of TM5 and TM6, consistent with the general mode of GPCR activation by ligand. (4)(5)(6)8,9,12). By combining data collected from extensive photoaffinity cross-linking studies with molecular modeling, a model of the PTH-PTHR1 bimolecular complex has been generated that identifies regions of contact at the interface between the hormone and the receptor.…”

Section: Molecular Modeling Of the Site Of Interaction Of Position 1 mentioning

confidence: 99%

“…The Cys1-PTH peptide ([Cys 1 ,Nle 8,18 , Arg 13,26,27 ,2-Nal 23 ,Tyr 34 ]bPTH-(1-34)-NH 2 ) was synthesized using Fmoc chemistry in our laboratory using a Symphony peptide synthesizer (Protein Technologies, Tucson, AZ) and purified using methods described previously (12). Cys was introduced as Fmoc-Cys (Trt) for protection during synthesis.…”

Section: Peptide Synthesis and Radioiodinationmentioning

confidence: 99%

Mapping Peptide Hormone−Receptor Interactions Using a Disulfide-Trapping Approach

et al. 2008

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Efforts to elucidate the nature of the bimolecular interaction of parathyroid hormone (PTH) with its cognate receptor, the PTH receptor type 1 (PTHR1), have relied heavily on benzoylphenylalanine-(Bpa-) based photoaffinity cross-linking. However, given the flexibility, size, and shape of Bpa, the resolution at the PTH-PTHR1 interface appears to be reaching the limit of this technique. Here we employ a disulfide-trapping approach developed by others primarily for use in screening compound libraries to identify novel ligands. In this method, cysteine substitutions are introduced into a specific site within the ligand and a region in the receptor predicted to interact with each other. Upon ligand binding, if these cysteines are in close proximity, they form a disulfide bond. Since the geometry governing disulfide bond formation is more constrained than Bpa cross-linking, this novel approach can be employed to generate a more refined molecular model of the PTH-PTHR1 complex. Using a PTH analogue containing a cysteine at position 1, we probed 24 sites and identified 4 in PTHR1 to which cross-linking occurred. Importantly, previous photoaffinity cross-linking studies using a PTH analogue with Bpa at position 1 only identified a single interaction site. The new sites identified by the disulfide-trapping procedure were used as constraints in molecular dynamics simulations to generate an updated model of the PTH-PTHR1 complex. Mapping by disulfide trapping extends and complements photoaffinity cross-linking. It is applicable to other peptide-receptor interfaces and should yield insights about yet unknown sites of ligand-receptor interactions, allowing for generation of more refined models.Class II G protein-coupled receptors (GPCRs) 1 interact with physiologically important peptides. There is intense study of how these peptides associate with their cognate receptors since elucidation of these interactions should provide important insights for the rational design of ligands with enhanced pharmacological properties for use in treating an array of diseases (1,2). Over the past decade, photoaffinity cross-linking has been employed to study the interaction of a number of peptide-GPCR interactions (3). This technique involves systematically probing the receptor for regions of interaction using a peptide that incorporates a photoreactive moiety that irreversibly cross-links to the receptor. We and others have used this approach extensively to study the interaction of parathyroid hormone (PTH) with its cognate receptor, the PTH receptor type 1 (PTHR1) (4-12). This hormone-receptor system plays an integral role in calcium metabolism and bone biology. † This work was supported in part by Grants DK47490 (to M.R.) and GM54082 (to D.F.M.) from the National Institutes of Health.* Address correspondence to this author. Phone: (617) Our research is focused on studying the bimolecular interface of the PTH-PTHR1 complex in order to gain insights that will aid the design of ligands of PTHR1 for the treatment of osteoporosis and o...

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Photoaffinity Cross-linking Identifies Differences in the Interactions of an Agonist and an Antagonist with the Parathyroid Hormone/Parathyroid Hormone-related Protein Receptor

Cited by 106 publications

References 49 publications

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

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

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

Mapping Peptide Hormone−Receptor Interactions Using a Disulfide-Trapping Approach

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