Glycosylation is important for binding to human calcitonin receptors

Human calcitonin receptor (hCTR) subtypes contain three or four potential A sn-linked glycosylation sires in their extracellular amino termini. The rol e of glycosylation in hCTR function has not been identified, but it has bee n suggested that inhibition of glycosylation does nor affect binding or sig naling. To determine the role of glycosylation in hCTR biology, we studied the effects of inhibition of glycosylation and of substitution of Asn resid ues that are potential glycosylation sites. Native and mutated hCTRs were s tudied after transient expression in monkey kidney COS-1 cells. Tunicamycin , administered as part of a treatment protocol that inhibited glycosylation of all expressed receptors, decreased salmon calcitonin (sCT) binding affi nities and signaling potencies at hCTRs with three or four potential glycos ylation sites. In hCTRS, which contains three potential glycosylation sites at positions 26, 78, and 83, site-specific substitution of Asn-26 by Ala h ad no effect on sCT binding affinity or potency, whereas substitution of As n-78 or Asn-83 lowered sCT affinity and potency. A mutant hCTR3 in which al l three Asn residues were substituted with Ala exhibited no high-affinity s CT binding and potencies of several calcitonin analogues that were more tha n 100-fold lower than that of native hCTR3, Our data show that glycosylatio n is important: for high-affinity binding and potency of calcitonin analogu es at hCTRs.