Agonist-promoted internalization of a ternary complex between calcitonin receptor-like receptor, receptor activity-modifying protein 1 (RAMP1), and beta-arrestin.
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.
Hilairet
S
S
Département de Biochimie, Université de Montréal, Montréal, Québec, H3C 3J7, Canada.
Bélanger
C
C
Bertrand
J
J
Laperrière
A
A
Foord
S M
SM
Bouvier
M
M
D019390:Arrestins / Q000502:physiology*; D015740:Calcitonin Gene-Related Peptide / Q000494:pharmacology; D058286:Calcitonin Receptor-Like Protein; D019281:Dimerization; D034281:Dynamins; D004705:Endocytosis; D020558:GTP Phosphohydrolases; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism*; D010766:Phosphorylation; D058262:Receptor Activity-Modifying Protein 1; D058261:Receptor Activity-Modifying Proteins; D018003:Receptors, Calcitonin / Q000378:metabolism*; D018015:Receptors, Calcitonin Gene-Related Peptide / Q000378:metabolism; D000071557:beta-Arrestins
D019390:Arrestins; C545578:CALCRL protein, human; D058286:Calcitonin Receptor-Like Protein; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C545502:RAMP1 protein, human; D058262:Receptor Activity-Modifying Protein 1; D058261:Receptor Activity-Modifying Proteins; D018003:Receptors, Calcitonin; D018015:Receptors, Calcitonin Gene-Related Peptide; D000071557:beta-Arrestins; D020558:GTP Phosphohydrolases; D034281:Dynamins; D015740:Calcitonin Gene-Related Peptide