Structure−Function Analysis of RAMP1 by Alanine Mutagenesis
- 12 December 2008
- journal article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 48 (1), 198-205
- https://doi.org/10.1021/bi801869n
Abstract
Receptor activity modifying protein 1 (RAMP1) is an integral component of several receptors including the calcitonin gene-related peptide (CGRP) receptor. It forms a complex with the calcitonin receptor-like receptor (CLR) and is required for receptor trafficking and ligand binding. The N-terminus of RAMP1 comprises three helices. The current study investigated regions of RAMP1 important for CGRP or CLR interactions by alanine mutagenesis. Modeling suggested the second and third helices were important in protein−protein interactions. Most of the conserved residues in the N-terminus (M48, W56, Y66, P85, N66, H97, F101, D113, P114, P115), together with a further 13 residues spread throughout three helices of RAMP1, were mutated to alanine and coexpressed with CLR in Cos 7 cells. None of the mutations significantly reduced RAMP expression. Of the nine mutants from helix 1, only M48A had any effect, producing a modest reduction in trafficking of CLR to the cell surface. In helix 2 Y66A almost completely abolished CLR trafficking; L69A and T73A reduced the potency of CGRP to produce cAMP. In helix 3, H97A abolished CLR trafficking; P85A, N86A, and F101A had caused modest reductions in CLR trafficking and also reduced the potency of CGRP on cAMP production. F93A caused a modest reduction in CLR trafficking alone and L94A increased cAMP production. The data are consistent with a CLR recognition site particularly involving Y66 and H97, with lesser roles for adjacent residues in helix 3. L69 and T73 may contribute to a CGRP recognition site in helix 2 also involving nearby residues.Keywords
This publication has 21 references indexed in Scilit:
- GPCR modulation by RAMPsPharmacology & Therapeutics, 2006
- Receptor-activity-modifying proteins are required for forward trafficking of the calcium-sensing receptor to the plasma membraneJournal of Cell Science, 2005
- Mechanisms of peptide and nonpeptide ligand binding to Class B G-protein-coupled receptorsDrug Discovery Today, 2005
- The Extracellular Domain of Receptor Activity-modifying Protein 1 Is Sufficient for Calcitonin Receptor-like Receptor FunctionJournal of Biological Chemistry, 2003
- Novel Receptor Partners and Function of Receptor Activity-modifying ProteinsPublished by Elsevier BV ,2003
- Multiple Ramp Domains Are Required for Generation of Amylin Receptor Phenotype from the Calcitonin Receptor Gene ProductBiochemical and Biophysical Research Communications, 2000
- Multiple Amylin Receptors Arise from Receptor Activity-Modifying Protein Interaction with the Calcitonin Receptor Gene ProductMolecular Pharmacology, 1999
- The Amino Terminus of Receptor Activity Modifying Proteins Is a Critical Determinant of Glycosylation State and Ligand Binding of Calcitonin Receptor-Like ReceptorMolecular Pharmacology, 1999
- An Amylin Receptor Is Revealed Following Co-Transfection of a Calcitonin Receptor with Receptor Activity Modifying Proteins-1 or -3Endocrinology, 1999
- RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptorNature, 1998