Role of N-Linked Glycosylation on the Function and Expression of the Human Secretin Receptor

Pang, Ronald Ting Kai; Ng, Sophia; Cheng, Christopher H.K.; Holtmann, Martin; Miller, Laurence J.; Chow, Billy Kwok Chong

doi:10.1210/endo.140.11.7134

Cited by 35 publications

(29 citation statements)

References 48 publications

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“…Our results show that modification of the C-terminal binding site by conversion of the peptide amide to the corresponding acid results in an analogue that exhibits a dramatic loss in affinity for receptor NT and full-length receptors as well, indicating involvement of the C-terminal Ucn1 (32)(33)(34)(35)(36)(37)(38)(39)(40) domain in binding to CRF receptor NT. It should be noted that this C-terminal modification does not result in a conformational change of the ligand as determined by CD measurements in FIGURE 4: Inhibition of specific [ 125 I-Tyr 0 ]-urocortin 1 binding to CRF-rNT by unlabeled CRF-like ligands urocortin 1 and astressin using the SPA technology: (A) data points for binding to CRF 1 -rNT represent pooled data from three independent experiments performed in triplicate for both ligands; (B) data points for binding to CRF 2(a) -rNT represent pooled data from six independent experiments (astressin) and nine independent experiments (urocortin 1) performed in triplicate.…”

Section: Discussionmentioning

confidence: 81%

Impact of N-Terminal Domains for Corticotropin-Releasing Factor (CRF) Receptor−Ligand Interactions

et al. 2005

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The large extracellular N-terminal domains (NTs) of class B G protein-coupled receptors serve as major ligand binding sites. However, little is known about the ligand requirements for interactions with these receptor domains. Recently, we have shown that the most potent CRF receptor agonist urocortin 1 (Ucn1) has two segregated receptor binding sites Ucn1(1-21) and Ucn1(32-40). For locating the receptor domains interacting with these two sites, we have investigated the binding of appropriate Ucn1 analogues to the receptor N-termini compared to the corresponding full-length receptors. For this purpose receptor NTs of CRF(rat) subtypes 1 and 2(alpha) without their signal sequences were overexpressed in Escherichia coli and folded in vitro. For CRF2(a)-rNT, which bears five cysteine residues (C2-C6), the disulfide arrangement C2-C5 and C4-C6 was found, leaving C3 free. This is consistent with the disulfide pattern of CRF1-rNT, which has six cysteines and in which C1 is paired with C3. Binding studies of N-terminally truncated or C-terminally modified Ucn1 analogues demonstrate that it is the C-terminal part, Ucn1(11-40), that binds to receptor NT, indicating a two-domain binding mechanism for Ucn binding to receptor NT. Since the binding of Ucn1 to the juxtamembrane domain has been shown to be segregated from binding to the receptor N-terminus [Hoare et al. (2004) Biochemistry 43, 3996-4011], a third binding domain should exist, probably comprising residues 8-10 of Ucn, which particularly contribute to a high-affinity binding to full-length receptors but not to receptor NT.

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Section: Discussionmentioning

confidence: 81%

Impact of N-Terminal Domains for Corticotropin-Releasing Factor (CRF) Receptor−Ligand Interactions

et al. 2005

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“…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. We have previously established the sites of glycosylation of the secretin receptor that are utilized (24).…”

Section: Resultsmentioning

confidence: 99%

“…CNBr cleavage of the secretin receptor results in fragments ranging in mass from 1 to 11 kDa, three of which contain sites of glycosylation (24). Shown is a representative autoradiograph of a 10% NuPAGE gel used to separate the products of CNBr cleavage of the secretin receptor labeled with the Bpa 26 probe.…”

Section: Figmentioning

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

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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“…The glycosylation of the amino-terminal domain of the CCK receptor is probably most responsible for this resistance to proteolysis. This is a recognized function of the glycosylation of membrane proteins (35,36). Another established function for membrane protein glycosylation relates to assisting in solubility and folding during biosynthesis that is also likely relevant to the CCK receptor.…”

Section: Discussionmentioning

confidence: 99%

Refinement of the Structure of the Ligand-occupied Cholecystokinin Receptor Using a Photolabile Amino-terminal Probe

Ding

Dolu²,

Hadac³

et al. 2001

Journal of Biological Chemistry

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Affinity labeling is a powerful tool to establish spatial approximations between photolabile residues within a ligand and its receptor. Here, we have utilized a cholecystokinin (CCK) analogue with a photolabile benzoylphenylalanine (Bpa) sited in position 24, adjacent to the pharmacophoric domain of this hormone (positions 27-33). This probe was a fully efficacious agonist that bound to the CCK receptor saturably and with high affinity (K i ‫؍‬ 8.9 ؎ 1.1 nM). It covalently labeled the CCK receptor either within the amino terminus (between Asn 10 and Lys 37 ) or within the third extracellular loop (Glu 345 ), as demonstrated by proteolytic peptide mapping, deglycosylation, micropurification, and Edman degradation sequencing. Truncation of the receptor to eliminate residues 1-30 had no detrimental effect on CCK binding, stimulated signaling, or affinity labeling through a residue within the pharmacophore (Bpa 29 ) but resulted in elimination of the covalent attachment of the Bpa 24 probe to the receptor. Thus, the distal amino terminus of the CCK receptor resides above the docked ligand, compressing the portion of the peptide extending beyond its pharmacophore toward the receptor core. Exposure of wild type and truncated receptor constructs to extracellular trypsin damaged the truncated construct but not the wild type receptor, suggesting that this domain also may play a protective role. Use of these additional insights into molecular approximations provided key constraints for molecular modeling of the peptide-receptor complex, supporting the counterclockwise organization of the transmembrane helical domains.Guanine nucleotide-binding protein (G protein) 1 -coupled receptors represent a remarkable group of structurally homologous membrane proteins that can bind and be activated by widely diverse ligands. The molecular details of how ligands as structurally dissimilar as photons, biogenic amines, peptides, and glycoproteins can elicit similar conformational changes in the cytosolic face of their receptors (where G protein-coupling occurs) are far from clear. Our best understanding of this process relates to the smallest ligands that appear to bind within the confluence of helices within the lipid bilayer (1), where we have analogous low resolution crystal structures on which to rely (2, 3). As the ligands get larger and more structurally complex, the binding domains tend to move toward the extracellular face of the membrane, with extracellular loop and amino-terminal tail domains becoming more important (1, 4). These are receptor domains for which we have minimal meaningful structural data.We have been quite interested in the molecular basis of ligand binding to the type A cholecystokinin (CCK) receptor (5-10). This receptor is a member of the class I family of G protein-coupled receptors, along with rhodopsin and the ␤-adrenergic receptor (11). CCK occurs as a series of linear peptides, having lengths ranging from 8 to 58 residues (12). These all share their carboxyl-terminal domain, with the carboxylterminal h...

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Role of N-Linked Glycosylation on the Function and Expression of the Human Secretin Receptor

Cited by 35 publications

References 48 publications

Impact of N-Terminal Domains for Corticotropin-Releasing Factor (CRF) Receptor−Ligand Interactions

Impact of N-Terminal Domains for Corticotropin-Releasing Factor (CRF) Receptor−Ligand Interactions

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

Refinement of the Structure of the Ligand-occupied Cholecystokinin Receptor Using a Photolabile Amino-terminal Probe

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