Aspartate(69) of the calcitonin-like receptor is required for its functional expression together with receptor-activity-modifying proteins 1 and -2.
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 Asp(69) (D(69)) is present in all family B1 G-protein-coupled receptors. Here the D(69) 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 EC(50) of 0.17 and 0.50 nM, 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 EC(50) of CGRP and AM were 1000-fold higher than those with the intact CLR. With the D69N mutant the EC(50) of CGRP was 0.48 nM and that of AM 0.44 nM, 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.
Ittner
Lars M
LM
Research Laboratory for Calcium Metabolism, Departments of Orthopedic Surgery and Medicine, University of Zurich, Klinik Balgrist, Forchstrasse 340, Zurich 8008, Switzerland.
Luessi
Felix
F
Koller
Daniela
D
Born
Walter
W
Fischer
Jan A
JA
Muff
Roman
R
D053607:Adrenomedullin; D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism*; D019556:COS Cells; D015740:Calcitonin Gene-Related Peptide / Q000378:metabolism; D002522:Chlorocebus aethiops; D000242:Cyclic AMP / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000378:metabolism; D051381:Rats; D058262:Receptor Activity-Modifying Protein 1; D058263:Receptor Activity-Modifying Protein 2; D058261:Receptor Activity-Modifying Proteins; D018003:Receptors, Calcitonin / Q000235:genetics* / Q000378:metabolism*; D016415:Sequence Alignment
D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D010455:Peptides; C545505:Ramp1 protein, mouse; C545506:Ramp2 protein, mouse; D058262:Receptor Activity-Modifying Protein 1; D058263:Receptor Activity-Modifying Protein 2; D058261:Receptor Activity-Modifying Proteins; D018003:Receptors, Calcitonin; D053607:Adrenomedullin; D001224:Aspartic Acid; D000242:Cyclic AMP; D015740:Calcitonin Gene-Related Peptide