RELATIONSHIP BETWEEN NATIVE AND RECOMBINANT CHOLECYSTOKININ RECEPTORS- ROLE OF DIFFERENTIAL GLYCOSYLATION

Citation
Em. Hadac et al., RELATIONSHIP BETWEEN NATIVE AND RECOMBINANT CHOLECYSTOKININ RECEPTORS- ROLE OF DIFFERENTIAL GLYCOSYLATION, Pancreas, 13(2), 1996, pp. 130-139
Citations number
42
Categorie Soggetti
Endocrynology & Metabolism",Physiology
Journal title
ISSN journal
08853177
Volume
13
Issue
2
Year of publication
1996
Pages
130 - 139
Database
ISI
SICI code
0885-3177(1996)13:2<130:RBNARC>2.0.ZU;2-R
Abstract
In an attempt to establish the relationship between the protein encode d by the recently cloned type A cholecystokinin (CCK) receptor cDNA an d the two distinct plasmalemmal proteins on the rat pancreatic acinar cell that were previously described as candidates to represent this re ceptor, we have established a Chinese hamster ovary (CHO) cell line st ably expressing large amounts of this recombinant protein and have use d biochemical methods to characterize it directly. Upon affinity label ing, this protein migrated faster on a sodium dodecyl sulfate-polyacry lamide gel than the M(r) 85,000-95,000 molecule previously felt to rep resent the best candidate. However, deglycosylation with endoglycosida se F demonstrated that it had the same size core protein as that candi date, and this identification was further supported by protease peptid e mapping, We postulated that the structural differences between the r ecombinant and the native proteins related to differences in glycosyla tion. Consistent with this, lectin-binding experiments demonstrated th at both represented complex glycoproteins but that only the native rec eptor-bound Ulex europeus agglutinin I. Since this lectin binds to fuc ose residues that are added late in glycoprotein biosynthesis, it is p ossible that the distinct processing observed affected only that step. In spite of this structural difference, the type A CCK receptor-beari ng CHO cell CCK receptor was functionally indistinguishable from the n ative acinar cell receptor. This included its ability to initiate sign aling cascades, its sensitivity to stable GTP analogues, and its bindi ng affinities for agonists and antagonists. The fidelity of this recep tor expression system, while representing a 25-fold increase in recept or density over the native pancreatic acinar cell, should provide an i deal substrate for the examination of structure-function relationships within this molecule.