TY - JOUR
T1 - Role of N-linked glycosylation on the function and expression of the human secretin receptor
AU - Pang, Ronald Ting Kai
AU - Ng, Samuel Sai Ming
AU - Cheng, Christopher Hon Ki
AU - Holtmann, Martin H.
AU - Miller, Laurence J.
AU - Chow, Billy Kwok Chong
N1 - Copyright:
Copyright 2018 Elsevier B.V., All rights reserved.
PY - 1999
Y1 - 1999
N2 - Secretin is a 27-amino acid long peptide hormone that regulates pancreatic water, bicarbonate, enzymes, and potassium ion secretion. The human secretin receptor (hSR) is a glycoprotein consisting of 440 amino acids, of which there are 5 putative N-linked glycosylation sites at positions Asn72, Asn100, Asn106, Asn128 (N-terminal ectodomain), and Asn291 (second exoloop). Through functional analysis of the hSR-transfected cells cultured in the presence of various glycosylation inhibitors, it was found that tunicamycin and castanospermine were able to significantly reduce the secretin-stimulated cAMP response. On the other hand, the effects of other inhibitors, swainsonine and deoxymannojirimycin, were much lower, suggesting that the high mannose-type carbohydrate side-chain is essential to the expression of a fully functional hSR. The role of individual N-linked glycosylation sites was studied by mutation analysis (Asn to Leu or Ser to Ala) coupled to measurements of cAMP accumulation and extracellular acidification rate. The ED50 values of the wild-type receptor in these two assay systems were 0.25 and 0.11 nM, respectively, and mutation at position 100, 106, or 291 did not affect either the ED50 values or the maximal responses in the two assays. However, the Asn72Leu and Ser74Ala mutations reduced the maximal responses and increased the ED50 values in both assays, suggesting that this site is a true glycosylation signal. This hypothesis was further supported by competitive binding studies, the same mutants were found to be defective in binding with [125I]secretin. To evaluate whether the change in receptor function of the mutants is caused by the change in the process of presenting the receptor to the cell surface, the mutants and the wild-type receptor were tagged with a c-Myc epitope at the C-termini. Using an anti-c-Myc monoclonal antibody and confocal microscopy, all of the mutant receptors were found to be expressed and delivered to the plasma membrane.
AB - Secretin is a 27-amino acid long peptide hormone that regulates pancreatic water, bicarbonate, enzymes, and potassium ion secretion. The human secretin receptor (hSR) is a glycoprotein consisting of 440 amino acids, of which there are 5 putative N-linked glycosylation sites at positions Asn72, Asn100, Asn106, Asn128 (N-terminal ectodomain), and Asn291 (second exoloop). Through functional analysis of the hSR-transfected cells cultured in the presence of various glycosylation inhibitors, it was found that tunicamycin and castanospermine were able to significantly reduce the secretin-stimulated cAMP response. On the other hand, the effects of other inhibitors, swainsonine and deoxymannojirimycin, were much lower, suggesting that the high mannose-type carbohydrate side-chain is essential to the expression of a fully functional hSR. The role of individual N-linked glycosylation sites was studied by mutation analysis (Asn to Leu or Ser to Ala) coupled to measurements of cAMP accumulation and extracellular acidification rate. The ED50 values of the wild-type receptor in these two assay systems were 0.25 and 0.11 nM, respectively, and mutation at position 100, 106, or 291 did not affect either the ED50 values or the maximal responses in the two assays. However, the Asn72Leu and Ser74Ala mutations reduced the maximal responses and increased the ED50 values in both assays, suggesting that this site is a true glycosylation signal. This hypothesis was further supported by competitive binding studies, the same mutants were found to be defective in binding with [125I]secretin. To evaluate whether the change in receptor function of the mutants is caused by the change in the process of presenting the receptor to the cell surface, the mutants and the wild-type receptor were tagged with a c-Myc epitope at the C-termini. Using an anti-c-Myc monoclonal antibody and confocal microscopy, all of the mutant receptors were found to be expressed and delivered to the plasma membrane.
UR - http://www.scopus.com/inward/record.url?scp=0033303551&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0033303551&partnerID=8YFLogxK
U2 - 10.1210/endo.140.11.7134
DO - 10.1210/endo.140.11.7134
M3 - Article
C2 - 10537138
AN - SCOPUS:0033303551
SN - 0013-7227
VL - 140
SP - 5102
EP - 5111
JO - Endocrinology
JF - Endocrinology
IS - 11
ER -