Visualization of the Calcitonin Receptor-like Receptor and Its Receptor Activity-modifying Proteins during Internalization and Recycling

Abstract

Expression of the calcitonin receptor-like receptor (CRLR) and its receptor activity modifying proteins (RAMPs) can produce calcitonin gene-related peptide (CGRP) receptors (CRLR/RAMP1) and adrenomedullin (AM) receptors (CRLR/RAMP2 or -3). A chimera of the CRLR and green fluorescent protein (CRLR-GFP) was used to study receptor localization and trafficking in stably transduced HEK 293 cells, with or without cotransfection of RAMPs. CRLR-GFP failed to generate responses to CGRP or AM without RAMPs. Furthermore, CRLR-GFP was not found in the plasma membrane and its localization was unchanged after agonist exposure. When stably coexpressed with RAMPs, CRLR-GFP appeared on the cell surface and was fully active in intracellular cAMP production and calcium mobilization. Agonist-mediated internalization of CRLR-GFP was observed in RAMP1/CGRP or AM, RAMP2/AM, and RAMP3/AM, which occurred with similar kinetics, indicating the existence of ligand-specific regulation of CRLR internalization by RAMPs. This internalization was strongly inhibited by hypertonic medium (0.45 M sucrose) and paralleled localization of rhodamine-labeled transferrin, suggesting that CRLR endocytosis occurred predominantly through a clathrin-dependent pathway. A significant proportion of CRLR was targeted to lysosomes upon binding of the ligands, and recycling of the internalized CRLR was not efficient. In HEK 293 cells stably expressing CRLR-GFP and Myc-RAMPs, these rhodamine-labeled RAMPs were co-localized with CRLR-GFP in the presence and absence of the ligands. Thus, the CRLR is endocytosed together with RAMPs via clathrin-coated vesicles, and both the internalized molecules are targeted to the degradative pathway.

Identification of the Human Receptor Activity-modifying Protein 1 Domains Responsible for Agonist Binding Specificity

“…Neither 10 nM h␣CGRP nor hAM elicited significant increases in cAMP in nontransfected HEK-293 cells or cells transfected with HA-hRAMP1 or hCRLR alone. On the other hand, the coexpression of hCRLR with HA-hRAMP1 enabled CGRP and AM to each elicit significant increases in cAMP, which is consistent with previous reports that CGRP and AM bind to the CRLR/RAMP1 heterodimer with similar affinities (8,9,12,24,25). Similar CGRPand AM-evoked increases in cAMP levels were seen when hCRLR was co-expressed with D74-76, D105-107, or D109-112.…”

“…The cells were then incubated for 4 h at 4°C with [ 125 I]h␣CGRP (100 pM) in the presence (for nonspecific binding) or absence (for total binding) of 1 M unlabeled h␣CGRP in modified Krebs-Ringer-HEPES medium (9). They were then washed twice with ice-cold PBS and harvested with 0.5 M NaOH, and the associated cellular radioactivity was measured in a ␥-counter.…”

“…To evaluate cell surface expression, cells were harvested following transient transfection, washed twice with PBS, resuspended in ice-cold FACS buffer (9), and then incubated with anti-HA-FITC antibody (1:50 dilution) for 60 min at 4°C in the dark. For evaluation of whole cell expression, cells were first permeabilized using IntraPrep TM reagents (Beckman Coulter, Fullerton, CA) according to the manufacturer's instructions and then incubated with anti-HA-FITC antibody (1:50 dilution) for 15 min at room temperature in the dark.…”

“…Co-expression of CRLR with RAMP1 leads to both proteins being presented at the plasma membrane as a heterodimeric CGRP receptor, whereas co-expression of CRLR with RAMP2 or RAMP3 enables the resultant heterodimer to function as a AM receptor (6,8,9). Upon binding their respective agonist, both receptors mediate a rise in intracellular cAMP levels as well as Ca 2ϩ mobilization and undergo internalization and intracellular trafficking with similar kinetics (9). One recent study (10) shows that the CRLR/RAMP1 heterodimer functions fully as both an AM and a CGRP receptor, which may explain why many actions of AM are potently antagonized by the CGRP receptor antagonist CGRP- (8 -37).…”

“…The three RAMP isoforms (RAMP1, RAMP2, and RAMP3), which share only 30% sequence identity and differ in their tissue distributions, are all comprised of ϳ160 amino acids that make up a large extracellular N-terminal domain, a single membrane-spanning domain, and a very short cytoplasmic domain (6, 7). Co-expression of CRLR with RAMP1 leads to both proteins being presented at the plasma membrane as a heterodimeric CGRP receptor, whereas co-expression of CRLR with RAMP2 or RAMP3 enables the resultant heterodimer to function as a AM receptor (6,8,9). Upon binding their respective agonist, both receptors mediate a rise in intracellular cAMP levels as well as Ca 2ϩ mobilization and undergo internalization and intracellular trafficking with similar kinetics (9).…”