Electrophoretic mobility and glycosylation characteristics of heterogeneously expressed calcitonin receptors.
The translated calcitonin receptor (CTR) complementary DNA sequences contain potential N-linked glycosylation sites within the extracellular N-terminus. We investigated the relative molecular mass (M(r)) and degree of N-linked glycosylation of five cloned CTRs (pig, rat C1a, rat C1b, human I1-ve, and human I1+ve), together with the pig hypothalamic CTR, to analyze the potential contribution of carbohydrate moieties to the molecular identity of these receptors. Receptors were cross-linked to 125I-salmon CT with the homobifunctional reagent bis(sulfosuccinimidyl) suberate. Autoradiographic analysis of the cross-linked receptors, following SDS-PAGE, revealed apparent M(r)S, ranging between 70,000 and 80,000 for the rat, human, and pig hypothalamic receptors. However, the cloned, expressed pig CTR was much smaller (approximately 58,000). The lower M(r) of the cloned pig CTR appeared to be due to absence of N-terminal residues, but this did not impact on ligand-receptor specificity when compared with the hypothalamic pig CTR. Cleavage under nondenaturing conditions of N-linked sugars from the CTRs using endoglycosidase F (Endo F), increased the electrophoretic mobility of all receptors, except the pig CTRs, by approximately 10 kDa. Under denaturing conditions, electrophoretic mobilities increased by approximately 30 kDa for the rat C1a, rat C1b, and humanI1-ve (expressed in human embryonic kidney-293 cells) CTRs and by approximately 20 kDa for the cloned pig, pig hypothalamic, and human CTR isoforms (expressed in baby hamster kidney cells). Competition binding studies using glycosylated and partially deglycosylated (nondenaturing conditions) receptor preparations demonstrated no significant differences in binding affinity or specificity. Thus the CTRs are N-linked glycoproteins whose degree of glycosylation is both cell-type and species dependent.
Quiza
M
M
Neurobiology Unit, St. Vincent's Institute of Medical Research, Fitzroy, Victoria, Australia.
Dowton
M
M
Perry
K J
KJ
Sexton
P M
PM
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D007031:Hypothalamus / Q000737:chemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D051381:Rats; D018003:Receptors, Calcitonin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013045:Species Specificity; D013552:Swine
D003432:Cross-Linking Reagents; D018003:Receptors, Calcitonin