Relationship between native and recombinant cholecystokinin receptors: Role of differential glycosylation

Elizabeth M. Hadac, Dilip V. Ghanekar, Eileen L. Holicky, Delia I. Pinon, Robert W. Dougherty, Laurence J. Miller

Research output: Contribution to journalArticle

97 Scopus citations

Abstract

In an attempt to establish the relationship between the protein encoded by the recently cloned type A cholecystokinin (CCK) receptor cDNA and the two distinct plasmalemmal proteins on the rat pancreatic acinar cell that were previously described as candidates to represent this receptor, we have established a Chinese hamster ovary (CHO) cell line stably expressing large amounts of this recombinant protein and have used biochemical methods to characterize it directly. Upon affinity labeling, this protein migrated faster on a sodium dodecyl sulfate-polyacrylamide gel than the M(r) 85,00095,000 molecule previously felt to represent the best candidate. However, deglycosylation with endoglycosidase F demonstrated that it had the same size core protein as that candidate, and this identification was further supported by protease peptide mapping. We postulated that the structural differences between the recombinant and the native proteins related to differences in glycosylation. Consistent with this, lectin-binding experiments demonstrated that both represented complex glycoproteins but that only the native receptor-bound Ulex europeus agglutinin I. Since this lectin binds to fucose residues that are added late in glycoprotein biosynthesis, it is possible that the distinct processing observed affected only that step. In spite of this structural difference, the type A CCK receptor-bearing CHO cell CCK receptor was functionally indistinguishable from the native acinar cell receptor. This included its ability to initiate signaling cascades, its sensitivity to stable GTP analogues, and its binding affinities for agonists and antagonists. The fidelity of this receptor expression system, while representing a 25-fold increase in receptor density over the native pancreatic acinar cell, should provide an ideal substrate for the examination of structure-function relationships within this molecule.

Original languageEnglish (US)
Pages (from-to)130-139
Number of pages10
JournalPancreas
Volume13
Issue number2
DOIs
StatePublished - Jan 1 1996

Keywords

  • Chinese hamster ovary (CHO) cell line
  • G protein-coupled receptor
  • Type A cholecystokinin receptor

ASJC Scopus subject areas

  • Internal Medicine
  • Endocrinology, Diabetes and Metabolism
  • Hepatology
  • Endocrinology

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