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

TitleRole of N-linked glycosylation on the function and expression of the human secretin receptor
Authors
Issue Date1999
PublisherThe Endocrine Society. The Journal's web site is located at http://endo.endojournals.org
Citation
Endocrinology, 1999, v. 140 n. 11, p. 5102-5111 How to Cite?
AbstractSecretin 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 Asn 72, Asn 100, Asn 106, Asn 128 (N-terminal ectodomain), and Asn 291 (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 ED 50 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 ED 50 values or the maximal responses in the two assays. However, the Asn 72Leu and Ser 74Ala mutations reduced the maximal responses and increased the ED 50 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.
Persistent Identifierhttp://hdl.handle.net/10722/84853
ISSN
2015 Impact Factor: 4.159
2015 SCImago Journal Rankings: 2.363
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorPang, RTKen_HK
dc.contributor.authorNg, SSMen_HK
dc.contributor.authorCheng, CHKen_HK
dc.contributor.authorHoltmann, MHen_HK
dc.contributor.authorMiller, LJen_HK
dc.contributor.authorChow, BKCen_HK
dc.date.accessioned2010-09-06T08:57:53Z-
dc.date.available2010-09-06T08:57:53Z-
dc.date.issued1999en_HK
dc.identifier.citationEndocrinology, 1999, v. 140 n. 11, p. 5102-5111en_HK
dc.identifier.issn0013-7227en_HK
dc.identifier.urihttp://hdl.handle.net/10722/84853-
dc.description.abstractSecretin 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 Asn 72, Asn 100, Asn 106, Asn 128 (N-terminal ectodomain), and Asn 291 (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 ED 50 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 ED 50 values or the maximal responses in the two assays. However, the Asn 72Leu and Ser 74Ala mutations reduced the maximal responses and increased the ED 50 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.en_HK
dc.languageengen_HK
dc.publisherThe Endocrine Society. The Journal's web site is located at http://endo.endojournals.orgen_HK
dc.relation.ispartofEndocrinologyen_HK
dc.rightsEndocrinology. Copyright © The Endocrine Society.en_HK
dc.titleRole of N-linked glycosylation on the function and expression of the human secretin receptoren_HK
dc.typeArticleen_HK
dc.identifier.openurlhttp://library.hku.hk:4550/resserv?sid=HKU:IR&issn=0013-7227&volume=140&spage=5102&epage=5111&date=1999&atitle=Role+of+N-Linked+Glycosylation+on+the+Function+and+Expression+of+the+Human+Secretin+Receptoren_HK
dc.identifier.emailPang, RTK: rtkpang@hku.hken_HK
dc.identifier.emailNg, SSM: ssmng@hku.hken_HK
dc.identifier.emailChow, BKC: bkcc@hku.hken_HK
dc.identifier.authorityPang, RTK=rp01761en_HK
dc.identifier.authorityNg, SSM=rp00767en_HK
dc.identifier.authorityChow, BKC=rp00681en_HK
dc.description.naturelink_to_subscribed_fulltext-
dc.identifier.pmid10537138-
dc.identifier.scopuseid_2-s2.0-0033303551en_HK
dc.identifier.hkuros47966en_HK
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-0033303551&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume140en_HK
dc.identifier.issue11en_HK
dc.identifier.spage5102en_HK
dc.identifier.epage5111en_HK
dc.identifier.isiWOS:000083200600025-
dc.publisher.placeUnited Statesen_HK
dc.identifier.scopusauthoridPang, RTK=7004376636en_HK
dc.identifier.scopusauthoridNg, SSM=7403358718en_HK
dc.identifier.scopusauthoridCheng, CHK=7404798014en_HK
dc.identifier.scopusauthoridHoltmann, MH=55394423200en_HK
dc.identifier.scopusauthoridMiller, LJ=7404986429en_HK
dc.identifier.scopusauthoridChow, BKC=7102826193en_HK

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