Em. Hadac et al., RELATIONSHIP BETWEEN NATIVE AND RECOMBINANT CHOLECYSTOKININ RECEPTORS- ROLE OF DIFFERENTIAL GLYCOSYLATION, Pancreas, 13(2), 1996, pp. 130-139
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.