The calcitonin-like receptor (CLR) associated with receptor-activity- modifying proteins (RAMP) 1 or -2 recognizes calcitonin gene-related peptide (CGRP) and adrenomedullin (AM), respectively. The amino acid sequence CNRTWDGWLCW corresponding to residues 64-74 in the extracellular N-terminus of the CLR is conserved. The Asp69 (D69) is present in all family B1 G-protein-coupled receptors. Here the D69 of a V5-tagged mouse CLR has been mutated to Ala (A), Glu (E), and Asn (N). The function of the intact and the mutant CLR was investigated in COS-7 cells coexpressing myc-tagged mouse RAMP1 or -2. In CLR/RAMP1 and -2 expressing cells CGRP and AM stimulated cAMP formation with an EC50 of 0.17 and 0.50nM, respectively. The expression of the D69A, D69E, and D69N mutants at the cell surface was comparable to that of the intact CLR. cAMP stimulation by CGRP and AM was abolished in the D69A mutant. With the D69E mutant the EC50 of CGRP and AM were 1000-fold higher than those with the intact CLR. With the D69N mutant the EC50 of CGRP was 0.48nM and that of AM 0.44nM, but the maximal cAMP formation was reduced to 24% and to 12% of cells with the intact CLR. Co-immunoprecipitation of RAMP1 with the CLR, indicating complex formation, was reduced with the D69A, D69N, and D69E mutants. RAMP2 co-precipitated with the mutant receptors indistinguishable from the intact CLR. In conclusion, mutation of D69 to N, E or A in the CLR did not affect its expression at the cell surface, but impaired or abolished the CGRP and AM receptor function in the presence of RAMP1 and -2, respectively.
|Number of pages||7|
|Journal||Biochemical and Biophysical Research Communications|
|Publication status||Published - 9 Jul 2004|
- Calcitonin gene-related peptide
- Calcitonin-like receptor
- Receptor mutations
- Receptor-activity-modifying proteins