Selective inactivation of adrenomedullin over calcitonin gene-related peptide receptor function by the deletion of amino acids 14-20 of the mouse calcitonin-like receptor.
The receptors for the neuropeptide calcitonin (CT) gene-related peptide (CGRP) and the multifunctional peptide hormone adrenomedullin (AM) are calcitonin-like receptor (CLR)/receptor-activity-modifying protein (RAMP) 1 and CLR/RAMP2 heterodimers, respectively. Here, the amino acid sequence TRNKIMT, corresponding to the residues 14-20 of the N terminus of the mouse (m) CLR, was found to be required for a functional mCLR/RAMP2 AM receptor. The deletion of amino acids 14-20 (Delta14-20) or their substitution by alanine (14-20A) did not affect the heterodimerization of the mCLR with mRAMP1 or mRAMP2, and the levels of expression at the surface of transiently transfected COS-7 cells were not altered. In mRAMP1/mCLR- or mRAMP1/mCLR-(Delta14-20)-expressing cells CGRP stimulated cAMP formation with EC(50) values of 0.12 +/- 0.01 and 1.5 +/- 0.4 nm, respectively. In mRAMP2/mCLR-expressing cells the EC(50) of AM was 0.8 +/- 0.2 nm. However, in cells expressing mRAMP2/mCLR-(Delta14-20) up to 10(-6) m AM failed to stimulate cAMP production. In mRAMP2/mCLR-(14-20A) expressing cells the cAMP response to AM was minimally restored, and the EC(50) was >100 nm. In conclusion, the deletion of the amino acid sequence TRNKIMT of the extreme N terminus of the mCLR maintained CGRP receptor function of mRAMP1/receptor heterodimers, but AM no longer activated the mutant mCLR-(Delta14-20) in the presence of mRAMP2. The TRNKIMT sequence is required for normal mCLR/mRAMP2 association, and as a consequence, high affinity AM binding signaling the activation of adenylyl cyclase.
Klinik Balgrist, Research Laboratory for Calcium Metabolism, Departments of Orthopedic Surgery and Medicine, University of Zurich, Forchstrasse 340, 8008 Zurich, Switzerland.
Daniela
D
Koller
Lars M
LM
Ittner
Roman
R
Muff
Knut
K
Husmann
Jan A
JA
Fischer
Walter
W
Born
D000262:Adenylyl Cyclases / Q000378:metabolism; D053607:Adrenomedullin; D000409:Alanine / Q000737:chemistry; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D015740:Calcitonin Gene-Related Peptide / Q000096:biosynthesis* / Q000235:genetics*; D058286:Calcitonin Receptor-Like Protein; D000242:Cyclic AMP / Q000378:metabolism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D017353:Gene Deletion; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000235:genetics* / Q000378:metabolism*; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D051381:Rats; D018003:Receptors, Calcitonin / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D014162:Transfection
D058286:Calcitonin Receptor-Like Protein; C545579:Calcrl protein, mouse; D010455:Peptides; D018003:Receptors, Calcitonin; D053607:Adrenomedullin; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; D015740:Calcitonin Gene-Related Peptide; D000409:Alanine