Direct Identification of a Second Distinct Site of Contact between Cholecystokinin and Its Receptor

Hadac, Elizabeth M.; Pinon, Delia I.; Ji, Zongshi; Holicky, Eileen L.; Henne, Randal M.; Lybrand, Terry P.; Miller, Laurence J.

doi:10.1074/jbc.273.21.12988

Cited by 79 publications

(157 citation statements)

References 22 publications

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“…Digestion of the intact receptor yielded a labeled M r ϭ 19,000 band that shifted to M r ϭ 10,000 after deglycosylation. This pattern of migration is identical to that we reported for labeling this receptor with both the Bpa 22 and Bpa 6 probes (1, 2). Considering the mass of the Bpa 26 probe (3, 341 Da) and this evidence of glycosylation of the labeled peptide, the first and third CNBr fragments of the secretin receptor are the best candidates to fit these data.…”

Section: Resultssupporting

confidence: 70%

“…This was the same position of the eluted [ 3 H]Leu that had been biosynthetically incorporated into this protein. 26 Probe-A cell line expressing the V16M-HA37 construct that had been characterized previously (2) was used to help localize the site of receptor labeling with the Bpa 22 probe. In addition, cells were prepared that expressed the V13M-HA37 secretin receptor mutant.…”

Section: Resultsmentioning

confidence: 99%

“…Nonspecific binding was determined in the presence of 1 M secretin and represented less than 20% of total bound radioactivity. 22 ]rat secretin-27 in the absence or presence of increasing concentrations of non-radioactive secretin (0 -1 M) prior to photolysis. This was performed for 30 min at 4°C in a Rayonet photochemical reactor (Southern New England Ultraviolet, Hamden, CT) equipped with 3500-Å lamps.…”

Section: Methodsmentioning

confidence: 99%

“…For this, purified radiolabeled receptor fragments were covalently coupled through their Cys residues to N-(2-aminoethyl-1)-3-aminopropyl glass beads (Sigma) using maleimidobenzoyl succinimide. This was followed by repetitive cycles of manual Edman degradation with quantitation of radioactivity released in each cycle (22).…”

Section: Affinity Labeling Studies-formentioning

confidence: 99%

“…Determination of Site of Covalent Attachment-After achieving definitive identification of the receptor fragment that was labeled with a distinct probe, its site of attachment was determined using radiochemical Edman degradation sequencing, as we previously reported (2,22). For this, purified radiolabeled receptor fragments were covalently coupled through their Cys residues to N-(2-aminoethyl-1)-3-aminopropyl glass beads (Sigma) using maleimidobenzoyl succinimide.…”

Section: Affinity Labeling Studies-formentioning

confidence: 99%

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

Dong

Asmann

Zang

et al. 2000

Journal of Biological Chemistry

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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...

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Section: Resultssupporting

confidence: 70%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Affinity Labeling Studies-formentioning

confidence: 99%

Section: Affinity Labeling Studies-formentioning

confidence: 99%

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

Dong

Asmann

Zang

et al. 2000

Journal of Biological Chemistry

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Structural characterization of peptide hormone/receptor interactions by NMR spectroscopy

Pellegrini

Mierke

1999

Biopolymers

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The structural characterization of peptide hormones and their interaction with G‐protein (guanine nucleotide‐binding regulatory protein) coupled receptors by high‐resolution nmr is described. The general approaches utilized can be categorized into three different classes based on their target: the ligand, the receptor, and the ligand/receptor complex. Examples of these different approaches, aimed at facilitating the rational design of peptides and peptidomimetics with improved pharmacological profiles, based on work carried out in our own laboratory, are given. In the ligand‐based approach, the high‐resolution structures of bradykinin analogues allowing for the development of a structure–activity relationship for activation of the B1 receptor are described. Studies targeting the receptor are to a large extent theoretical, based on computational molecular modeling. However, experimentally based structural features provided by high‐resolution nmr can be used to great advantage, providing insight into the mechanism of receptor function, as illustrated here with results from parathyroid hormone. A similar combination of theoretical methods, supplemented by high‐resolution structures from nmr has been utilized to probe the formation and stabilization of the ligand/receptor complex both for parathyroid hormone and cholecystokinin. In each of these three approaches, the importance of well‐designed peptide mimetics and accurate structural analysis by high‐resolution nmr, will be highlighted. © 1999 John Wiley & Sons, Inc. Biopoly 51: 208–220, 1999

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Peptide/benzodiazepine hybrids as ligands of CCKA and CCKB receptors

Escherich¹,

Lutz²,

Escrieut³

et al. 2000

Biopolymers

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Direct Identification of a Second Distinct Site of Contact between Cholecystokinin and Its Receptor

Cited by 79 publications

References 22 publications

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

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

Structural characterization of peptide hormone/receptor interactions by NMR spectroscopy

Peptide/benzodiazepine hybrids as ligands of CCKA and CCKB receptors

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