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

Department of Zoology, The University of Hong Kong (R.T.-K.P., S.S.-M.N., B.K.-C.C.), and the Department of Biochemistry, Chinese University of Hong Kong (C.H.-K.C.), Hong Kong, Special Administrative Region, Peoples Republic of China; and the Center for Basic Research in Digestive Diseases, Mayo Clinic and Foundation (M.H.H., L.J.M.), Rochester, Minnesota 55905

Address all correspondence and requests for reprints to: Dr. B. K. C. Chow, Department of Zoology, University of Hong Kong, Pokfulam Road, Hong Kong, Special Administrative Region, Peoples Republic of China. E-mail: bkcc{at}hkusua.hku.hk

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.

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