Distinct receptor activity-modifying protein domains differentially modulate interaction with calcitonin receptors.
Calcitonin receptors (CTRs) dimerize with receptor activity-modifying proteins (RAMPs) to generate high-affinity amylin (AMY) receptors; however, the relative contribution of individual RAMP domains to the formation of AMY receptors is poorly understood. We have used chimeras between RAMP1 and RAMP2 that specifically exchanged the N-terminal, transmembrane, or C-terminal domain and examined these in assays of [(125)I]amylin binding or peptide-induced cAMP signaling in COS-7 cells transiently transfected with wild-type or chimeric RAMPs and human CTRa. The specificity of peptides in competition for [(125)I]amylin binding was principally dictated by the N-terminal domain present in the chimeras; however, the maximal level of binding induced was dictated by the transmembrane domain present. This extended previous data (Zumpe et al., 2000) to provide a distinction between the transmembrane domain and the C terminus in this function. In contrast to the effects on binding, each of the RAMP domains played a role in the signaling phenotype of the receptors. In particular, the potency of calcitonin gene-related peptide (CGRP) was most influenced by the C-terminal domain present, in which the presence of the RAMP1 C-terminal domain led to increased potency over CTRa alone, whereas chimeras with the RAMP2 C-terminal domain did not induce increased CGRP potency. The data provide additional support for the importance of the N terminus in determining binding affinity but reveal a prominent role of the transmembrane domain in the strength of amylin binding and a unique role for the C terminus in signaling by peptides to stimulate cAMP production.
Department of Pharmacology, Building 13E, Monash University, Clayton, 3800 Victoria, Australia.
Madhara
M
Udawela
George
G
Christopoulos
Nanda
N
Tilakaratne
Arthur
A
Christopoulos
Anthony
A
Albiston
Patrick M
PM
Sexton
D000818:Animals; D019556:COS Cells; D015740:Calcitonin Gene-Related Peptide / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D000242:Cyclic AMP / Q000378:metabolism; D019281:Dimerization; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D058262:Receptor Activity-Modifying Protein 1; D058263:Receptor Activity-Modifying Protein 2; D058261:Receptor Activity-Modifying Proteins; D018003:Receptors, Calcitonin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D058287:Receptors, Islet Amyloid Polypeptide; D018000:Receptors, Peptide / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D008565:Membrane Proteins; D010455:Peptides; C545502:RAMP1 protein, human; C545503:RAMP2 protein, human; D058262:Receptor Activity-Modifying Protein 1; D058263:Receptor Activity-Modifying Protein 2; D058261:Receptor Activity-Modifying Proteins; D018003:Receptors, Calcitonin; D058287:Receptors, Islet Amyloid Polypeptide; D018000:Receptors, Peptide; D011993:Recombinant Fusion Proteins; D000242:Cyclic AMP; D015740:Calcitonin Gene-Related Peptide