Structure-function analysis of RAMP1-RAMP3 chimeras.
The role of receptor activity modifying protein 1 (RAMP1) in forming receptors with the calcitonin receptor-like receptor (CLR) and the calcitonin receptor (CTR) was examined by producing chimeras between RAMP1 and RAMP3. RAMPs have three extracellular helices. Exchange of helix 1 of the RAMPs or residues 62-69 in helix 2 greatly reduced CLR trafficking (a marker for CLR association). Modeling suggests that these exchanges alter the CLR recognition site on RAMP1, which is more exposed than on RAMP3. Exchange of residues 86-89 of RAMP1 had no effect on the trafficking of CLR but reduced the potency of human (h) alphaCGRP and adrenomedullin. However, these alterations to RAMP1 had no effect on the potency of hbetaCGRP. These residues of RAMP1 lie at the junction of helix 3 and its connecting loop with helix 2. Modeling suggests that the loop is more exposed in RAMP1 than RAMP3; it may play an important role in peptide binding, either directly or indirectly. Exchange of residues 90-94 of RAMP1 caused a modest reduction in CLR expression and a 15-fold decrease in CGRP potency. It is unlikely that the decrease in expression is enough to explain the reduction in potency, and so these may have dual roles in recognizing CLR and CGRP. For CTR, only 6 out of 26 chimeras covering the extracellular part of RAMP1 did not reduce agonist potency. Thus the association of CTR with RAMP1 seems more sensitive to changes in RAMP1 structure induced by the chimeras than is CLR.
Qi
Tao
T
School of Biological Sciences, University of Auckland, Auckland, New Zealand.
Simms
John
J
Bailey
Richard J
RJ
Wheatley
Mark
M
Rathbone
Dan L
DL
Hay
Debbie L
DL
Poyner
David R
DR
D053607:Adrenomedullin / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D058286:Calcitonin Receptor-Like Protein; D002460:Cell Line; D002522:Chlorocebus aethiops; D017124:Conserved Sequence; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D051381:Rats; D058262:Receptor Activity-Modifying Protein 1; D058264:Receptor Activity-Modifying Protein 3; D058261:Receptor Activity-Modifying Proteins; D018003:Receptors, Calcitonin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D014162:Transfection
C545578:CALCRL protein, human; D058286:Calcitonin Receptor-Like Protein; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C545502:RAMP1 protein, human; C545504:RAMP3 protein, human; D058262:Receptor Activity-Modifying Protein 1; D058264:Receptor Activity-Modifying Protein 3; D058261:Receptor Activity-Modifying Proteins; D018003:Receptors, Calcitonin; D011993:Recombinant Fusion Proteins; D053607:Adrenomedullin