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Glycosylation Is Important for Binding to Human Calcitonin Receptors

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Division of Molecular Medicine, Department of Medicine, Cornell University Medical College and The New York Hospital, New York, New York 10021
Cite this: Biochemistry 1999, 38, 6, 1866–1872
Publication Date (Web):January 21, 1999
https://doi.org/10.1021/bi981195e
Copyright © 1999 American Chemical Society

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    Abstract

    Human calcitonin receptor (hCTR) subtypes contain three or four potential Asn-linked glycosylation sites in their extracellular amino termini. The role of glycosylation in hCTR function has not been identified, but it has been suggested that inhibition of glycosylation does not affect binding or signaling. To determine the role of glycosylation in hCTR biology, we studied the effects of inhibition of glycosylation and of substitution of Asn residues that are potential glycosylation sites. Native and mutated hCTRs were studied 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 affinities and signaling potencies at hCTRs with three or four potential glycosylation sites. In hCTR3, which contains three potential glycosylation sites at positions 26, 78, and 83, site-specific substitution of Asn-26 by Ala had no effect on sCT binding affinity or potency, whereas substitution of Asn-78 or Asn-83 lowered sCT affinity and potency. A mutant hCTR3 in which all three Asn residues were substituted with Ala exhibited no high-affinity sCT binding and potencies of several calcitonin analogues that were more than 100-fold lower than that of native hCTR3. Our data show that glycosylation is important for high-affinity binding and potency of calcitonin analogues at hCTRs.

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     This work was supported by U.S. Public Health Service Grants DK43036 (to M.C.G.) and DK50673 (to D.R.N.).

    *

     To whom correspondence should be addressed:  Cornell University Medical College, 1300 York Ave., Room A328, New York, NY 10021. Telephone:  (212) 746-6275. Fax:  (212) 746-6289. E-mail:  mcgersh@ mail.med.cornell.edu.

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