Post-endocytic Sorting of Calcitonin Receptor-like Receptor and Receptor Activity-modifying Protein 1

Abstract

Calcitonin gene-related peptide (CGRP) 2 belongs to the calcitonin family of regulatory peptides and is produced by tissuespecific alternate splicing of transcripts from the calcitonin gene (1). CGRP is a potent vasodilator and mediator of neurogenic inflammation and pain transmission (2, 3). Notably, CGRP has a causative role in migraine headaches and is thus a mediator of human disease (4). In view of the importance of CGRP in health and disease, it is of interest to understand the mechanisms that control cellular responses to this peptide.Unusually for neuropeptides, the CGRP receptor is a heterodimer composed of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1). CLR is a G-protein-coupled receptor (GPCR) that shares 55% amino acid sequence identity with the calcitonin receptor (5), whereas RAMP1, RAMP2, and RAMP3 are single transmembrane proteins with ϳ30% identity (6). CLR functions as either a CGRP receptor, when coexpressed with RAMP1, or an adrenomedullin receptor, when coexpressed with RAMP2 or -3 (6); the extracellular domain of the RAMP imparts this specificity (7,8). When expressed alone, CLR is retained in the endoplasmic reticulum (6), and RAMP1 is retained in the endoplasmic reticulum and the Golgi apparatus (9 -11). However, when coexpressed, CLR and RAMP1 traffic to the plasma membrane. Thus, RAMP1 is also a chaperone that targets CLR to the plasma membrane. However, it remains to be determined if CLR and RAMP1 are invariably associated after receptor activation.Upon activation with CGRP, CLR, but not RAMP1, is phosphorylated and interacts with ␤-arrestins (12). ␤-Arrestins are adapters for clathrin and AP2, and the CLR, RAMP1, and ␤-arrestin complex undergoes dynamin-dependent endocytosis in clathrin-coated pits by well defined mechanisms (11, 12). However, little is known about the mechanisms of post-endocytic sorting of GPCRs, such as CLR, or accessory proteins, such as RAMP1, to degradative or recycling pathways. It is important to understand the post-endocytic sorting of CLR and RAMP1, because recycling may permit rapid resensitization of CGRP signaling, whereas degradation would prevent sustained, uncontrolled CGRP signaling during conditions of sustained peptide release.␤-Arrestins can influence the rate of recycling of GPCRs. "Class A" receptors (e.g. ␤ 2 adrenergic receptor (␤ 2 AR), -opi-* This work was supported by National Institutes of Health Grants DK39957 (to N. W. B.), DK43207 (to N. W. B.), DK57840 (to N. W. B.), and DK52388 (to E. F. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. RAMP1, receptor activity-modifying protein; NK 1 R, neurokinin 1 receptor; ␤ 2 AR, ␤ 2 adrenergic receptor; PAR 2 , protease-activated receptor 2; LAMP1, lysosomal-associated glycomembrane protein 1; EEA1, early endosomal antigen 1; BSA, bovine serum albumin; HA, hemagglutinin; ...

Endosomal Endothelin-converting Enzyme-1

“…PD-069185 (19) was a gift (Pfizer). Other reagents were from Sigma or have been described (12,13,20).…”

“…Measurement of [Ca 2ϩ ] i -[Ca 2ϩ ] i was measured in populations of cells using Fura2-AM (Invitrogen) as described (12,20). To assess resensitization, cells were incubated with vehicle or SP (10 nM, 0 or 10 min, 37°C), washed, and recovered in SP-free medium for 2 h. Cells were challenged with SP (10 nM), and [Ca 2ϩ ] i was measured.…”

“…Transfected Cells and Cell Lines-Generation and maintenance of human embryonic kidney 293 (HEK) FLP cells (Invitrogen) and HEKFLP stably expressing rat NK 1 R have been described (12,13,20). In some experiments cells were transiently transfected with rat NK 1 R, rat FLAG-NK 1 R⌬325-407, rat ␤-arrestin1 V53D, rat ␤-arrestin1 319 -418 , human ␤-arrestin2-GFP, human ECE-1c-GFP, or empty vector (control) using Lipofectamine2000 (Invitrogen).…”

“…To examine the interaction of NK 1 R⌬325-407 and ␤-arrestin2, NK 1 R⌬325-407 was labeled at the cell surface by incubating HEK-NK 1 R⌬325-407-␤-arrestin2-GFP cells with anti-FLAG for 30 min at 37°C (20). Cells were washed with PBS, stimulated with SP (10 nM, 10 min), washed, recovered in SP-free medium, and fixed in 4% paraformaldehyde, 100 mM PBS, pH 7.4 (20 min, 4°C).…”

“…Membranes were incubated with antibodies to pERK2 (E-4; 1:1,000), ERK2 (C-14; 1:5,000), c-Fos (9F6, 1:2,000), ECE-1 (1:1,000), EEA1 (1:10,000), Rab5a (1:2,000), LAMP1 (1:2,000), Na ϩ -K ϩ -ATPase (1:5,000), histone H3 (1:20,000), NK 1 R (1:10,000), ␤-arrestin2 (1:5,000), Src (1:1,000), Nur77 (1:200), phosphothreonine (1:1,000), or ␤-actin (A-5441, 1:10,000) overnight at 4°C. Membranes were treated with secondary or tertiary antibodies and analyzed with an Odyssey Infrared Imaging System (Li-COR Biosciences) (12,13,20).…”