The calcitonin receptor-like receptor (CRLR) requires heterodimerization with receptor activity modifying protein 1, RAMP-1, for cell surface expression and calcitonin gene-related peptide (CGRP) affinity. CRLR has three consensus sites for N-linked glycosylation. CRLR is a core glycosylated intracellular protein that becomes a mature glycosylated cell surface receptor in the presence of RAMP-1. The contribution of each of the N-linked glycosylation sites to CRLR glycosylation, cell surface expression, CGRP affinity and signal transduction was investigated. In addition to tunicamycin inhibition of N-linked glycosylation, site directed mutagenesis was used to construct seven mutants of HA epitope-tagged CRLR with C-terminal fusion to GFP (CRLR-GFP) each containing individual or combinations of Thr to Ala substitutions within consensus sites (Asn-X-Thr). Receptors were expressed alone or with RAMP-1 in tsA 201, HEK-293 cells. In the absence of RAMP-1, CRLR-GFP was equally glycosylated at each site and expressed as both a core glycosylated intracellular protein of Mr 70,000 and a mature cell surface receptor of Mr 82,000. CRLR-GFP co-expressed with RAMP-1, was expressed only as a mature glycosylated Mr 75,000 cell surface receptor as a result of glycosylation only at Asn66 and Asn118 and not at Asn123. Loss of glycosylation at either Asn66 or Asn118 had no significant effect on CGRP affinity or stimulated cAMP accumulation, while cell surface expression was reduced. Loss of glycosylation at both Asn66 and Asn118 decreased CGRP affinity and potency (but not efficacy) for increasing cAMP and further reduced cell surface expression. Tunicamycin inhibition caused a less severe reduction in CRLR-GFP function than site-directed mutagenesis and suggests that the amino acid substitutions themselves contribute to loss of function. RAMP-1 specific inhibition of N-linked glycosylation of CRLR at Asn123 and not Asn118 as well as the loss of CGRP affinity with the T125A substitution suggests that the area around Asn123 is a region of protein-protein interaction between RAMP-1 and CRLR. Digestion of CRLR-GFP with O-glycosidase revealed the presence O-linked glycosylation that may contribute to native CRLR function and/or compensate for experimentally induced loss in N-linked glycosylation.
