Mutations of the asparagine(117) residue of a receptor activity-modifying protein 1-dependent human calcitonin gene-related peptide receptor result in selective loss of function

The initially orphan human calcitonin (CT) receptor-like receptor (hCRLR) i nteracts with novel accessory receptor activity-modifying protein 1 (RAMP1) to reveal a functional CT gene-related peptide (CGRP) receptor. In mammali an cells, RAMP1 is required for mature N-glycosylation of the hCRLR predict ed to occur at Asn(60), Asn(112), and/or Asn(117) in the amino-terminal ext racellular domain. Here we have shown that the substitution of Asn(117) wit h Ala, Gin, Thr, or Pro abolished CGRP-evoked cAMP formation which was left unchanged when the Asn(117) was replaced with Asp. Moreover, the hCRLR and the Asn(117) mutants exhibited comparable N-glycosylation and cell surface expression, and the association with RAMP1 was only slightly impaired. In contrast, the hCRLR Asn(60,112) to Thr double mutant exhibited defective RA MP1-dependent N-glycosylation, and impaired cell surface expression and CGR P receptor function. Unlike Asn(60) and Asn(112), Asn(117) is normally not N-glycosylated, but essential for CGRP binding to the hCRLR-RAMP1 complex.