Abstract
The calcitonin receptor-like receptor (CRLR) is a seven-transmembrane domain (7TM) protein that requires the receptor activity-modifying protein 1 (RAMP1) to be expressed at the cell surface as a functional calcitonin gene-related peptide (CGRP) receptor. Although dimerization between the two molecules is well established, very little is known concerning the trafficking of this heterodimer upon receptor activation. Also, the subcellular localization and biochemical state of this ubiquitously expressed protein, in the absence of CRLR, remains poorly characterized. Here we report that when expressed alone RAMP1 is retained inside the cells where it is found in the endoplasmic reticulum and the Golgi predominantly as a disulfide-linked homodimer. In contrast, when expressed with CRLR, it is targeted to the cell surface as a 1:1 heterodimer with the 7TM protein. Although heterodimer formation does not involve intermolecular disulfide bonds, RAMP-CRLR association promotes the formation of intramolecular disulfide bonds within RAMP1. CGRP binding and receptor activation lead to the phosphorylation of CRLR and the internalization of the receptor as a stable complex. The internalization was found to be both dynamin- and beta-arrestin-dependent, indicating that the formation of a ternary complex between CRLR, RAMP1, and beta-arrestin leads to clathrin-coated pit-mediated endocytosis. These results therefore indicate that although atypical by its heterodimeric composition and its targeting to the plasma membrane, the CGRP receptor shares endocytotic mechanisms that are common to most classical 7TM receptors.
Highlights
The calcitonin family of regulatory peptides [1,2,3]
Given that at least one of the RAMPs is expressed in all tissues tested to date and that they are expressed independently of calcitonin-receptor like receptor (CRLR) [1,2,3, 5, 12], a better characterization of the subcellular localization of RAMP could shed some light on additional roles that could be played by these proteins
RAMP 2 and 3 were found in the lysosome following a treatment of 30 min with adrenomedullin [10], but whether the CRLR1⁄7RAMP complexes are endocytosed as a stable complex remains to be determined
Summary
Labeling in Permeabilized Cells—HEK293T cells stably expressing HA-CRLR or myc-RAMP1 were fixed with 3% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min and permeabilized for 10 min with 0.25% Triton X-100 in blocking buffer (PBS containing 0.2% BSA). The coverslips were washed in blocking buffer and incubated in the same buffer with the appropriate secondary antibodies (Texas Red-conjugated secondary antibodies for visualization of mycRAMP1 or HA-CRLR and Oregon Green-conjugated secondary antibodies for visualization of p58K or calnexin) at room temperature for 30 min in the dark. To determine cell-surface expression of both proteins, cells were incubated in DMEM/Hepes blocking buffer (20 mM Hepes, pH 7.4, 0.2% BSA in DMEM) for 30 min on ice and incubated for 1 h with primary antibody (rabbit polyclonal anti-myc and/or rat monoclonal anti-HA) diluted in EXPERIMENTAL PROCEDURES. Oregon Green-conjugated and Texas Red-conjugated secondary antibodies were purchased from Molecular Probes Inc. All other reagents were analytical grade and obtained from various commercial suppliers
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