Electrophoretic mobility and glycosylation characteristics of heterogeneously expressed calcitonin receptors

Maribel Quiza, Mark Dowton, Katie J. Perry, Patrick M. Sexton

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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(11- ve), and human(11+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 125 I-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 (~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 10 kDa. Under denaturing conditions, electrophoretic mobilities increased by ~30 kDa for the rat C1a, rat C1b, and human(11-ve) (expressed in human embryonic kidney-293 cells) CTRs and by ~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.

Original languageEnglish
Pages (from-to)530-539
Number of pages10
Issue number2
Publication statusPublished - 1 Jan 1997
Externally publishedYes

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