The N-terminal extracellular domain 23-60 of the calcitonin receptor-like receptor in chimeras with the parathyroid hormone receptor mediates association with receptor activity-modifying protein 1.
The calcitonin receptor-like receptor (CLR) requires the associated receptor activity-modifying protein (RAMP)1 to reveal a calcitonin gene-related peptide (CGRP) receptor. Here, the subdomain of the CLR that associates with RAMP1 has been identified in chimeras between the CLR and the parathyroid hormone (PTH) receptor 1 (PTHR). The PTHR alone does not interact with RAMP1. RAMP1 requires the CLR for its transport to the cell surface. Thus, receptor-dependent RAMP1 delivery to the plasma membrane and coimmunoprecipitation from the cell surface were used as measures for receptor/RAMP1 interaction. Several chimeric CLR-PTHR included the N-terminal amino acids 23-60 of the CLR transported RAMP1 to the surface of COS-7 cells much like the intact CLR. Moreover, RAMP1 coimmunoprecipitated with these receptors from the cell surface. A CLR deletion mutant, consisting of the N-terminal extracellular domain, the first transmembrane domain, and the C-terminal intracellular region, revealed the same results. Cyclic AMP was stimulated by CGRP in CLR/RAMP1 expressing cells (58 +/- 19-fold, EC(50) = 0.12 +/- 0.03 nM) and by PTH-related protein in cells expressing the PTHR (50 +/- 10-fold, EC(50) = 0.25 +/- 0.03 nM) or a PTHR with the N-terminal amino acids 23-60 of the CLR (23 +/- 5-fold, EC(50) > 1000 nM). Other chimeric CLR-PTHR were inactive. In conclusion, structural elements in the extreme N-terminus of the CLR between amino acids 23-60 are required and sufficient for CLR/RAMP1 cotransport to the plasma membrane and heterodimerization.
Ittner
Lars M
LM
Research Laboratory for Calcium Metabolism, Department of Orthopedic Surgery, University of Zurich, Balgrist University Hospital, Forchstrasse 340, 8008 Zurich, Switzerland.
Koller
Daniela
D
Muff
Roman
R
Fischer
Jan A
JA
Born
Walter
W
D000595:Amino Acid Sequence; D000818:Animals; D001693:Biological Transport, Active; D019556:COS Cells; D058286:Calcitonin Receptor-Like Protein; D002462:Cell Membrane / Q000378:metabolism; D002645:Chickens; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D051381:Rats; D058262:Receptor Activity-Modifying Protein 1; D058261:Receptor Activity-Modifying Proteins; D018003:Receptors, Calcitonin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018016:Receptors, Parathyroid Hormone / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid
D058286:Calcitonin Receptor-Like Protein; C545579:Calcrl protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C545505:Ramp1 protein, mouse; D058262:Receptor Activity-Modifying Protein 1; D058261:Receptor Activity-Modifying Proteins; D018003:Receptors, Calcitonin; D018016:Receptors, Parathyroid Hormone; D011993:Recombinant Fusion Proteins