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.
Koller
Daniela
D
Klinik Balgrist, Research Laboratory for Calcium Metabolism, Departments of Orthopedic Surgery and Medicine, University of Zurich, Forchstrasse 340, 8008 Zurich, Switzerland.
Ittner
Lars M
LM
Muff
Roman
R
Husmann
Knut
K
Fischer
Jan A
JA
Born
Walter
W
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