Role of N-linked glycosylation on the function and expression of the human secretin receptor

Ronald Ting Kai Pang, Samuel Sai Ming Ng, Christopher Hon Ki Cheng, Martin H. Holtmann, Laurence J Miller, Billy Kwok Chong Chow

Research output: Contribution to journalArticle

33 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)5102-5111
Number of pages10
JournalEndocrinology
Volume140
Issue number11
StatePublished - 1999

Fingerprint

Glycosylation
Secretin
Mutation
Swainsonine
Amino Acids
Tunicamycin
Competitive Binding
Peptide Hormones
Cell Surface Receptors
Mannose
Confocal Microscopy
Epitopes
Cultured Cells
Glycoproteins
Monoclonal Antibodies
Carbohydrates
Cell Membrane
Ions
secretin receptor
Water

ASJC Scopus subject areas

  • Endocrinology
  • Endocrinology, Diabetes and Metabolism

Cite this

Pang, R. T. K., Ng, S. S. M., Cheng, C. H. K., Holtmann, M. H., Miller, L. J., & Chow, B. K. C. (1999). Role of N-linked glycosylation on the function and expression of the human secretin receptor. Endocrinology, 140(11), 5102-5111.

Role of N-linked glycosylation on the function and expression of the human secretin receptor. / Pang, Ronald Ting Kai; Ng, Samuel Sai Ming; Cheng, Christopher Hon Ki; Holtmann, Martin H.; Miller, Laurence J; Chow, Billy Kwok Chong.

In: Endocrinology, Vol. 140, No. 11, 1999, p. 5102-5111.

Research output: Contribution to journalArticle

Pang, RTK, Ng, SSM, Cheng, CHK, Holtmann, MH, Miller, LJ & Chow, BKC 1999, 'Role of N-linked glycosylation on the function and expression of the human secretin receptor', Endocrinology, vol. 140, no. 11, pp. 5102-5111.
Pang RTK, Ng SSM, Cheng CHK, Holtmann MH, Miller LJ, Chow BKC. Role of N-linked glycosylation on the function and expression of the human secretin receptor. Endocrinology. 1999;140(11):5102-5111.
Pang, Ronald Ting Kai ; Ng, Samuel Sai Ming ; Cheng, Christopher Hon Ki ; Holtmann, Martin H. ; Miller, Laurence J ; Chow, Billy Kwok Chong. / Role of N-linked glycosylation on the function and expression of the human secretin receptor. In: Endocrinology. 1999 ; Vol. 140, No. 11. pp. 5102-5111.
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