Article

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

Department of Surgery, University of California, San Francisco, California 94143-0660, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 04/2007; 282(16):12260-71. DOI: 10.1074/jbc.M606338200
Source: PubMed

ABSTRACT Calcitonin receptor-like receptor (CLR) and the receptor activity-modifying protein 1 (RAMP1) comprise a receptor for calcitonin gene-related peptide (CGRP). Although CGRP induces endocytosis of CLR/RAMP1, little is known about post-endocytic sorting of these proteins. We observed that the duration of stimulation with CGRP markedly affected post-endocytic sorting of CLR/RAMP1. In HEK and SK-N-MC cells, transient stimulation (10(-7) M CGRP, 1 h), induced CLR/RAMP1 recycling with similar kinetics (2-6 h), demonstrated by labeling receptors in living cells with antibodies to extracellular epitopes. Recycling of CLR/RAMP1 correlated with resensitization of CGRP-induced increases in [Ca(2+)](i). Cycloheximide did not affect resensitization, but bafilomycin A(1), an inhibitor of vacuolar H(+)-ATPases, abolished resensitization. Recycling CLR and RAMP1 were detected in endosomes containing Rab4a and Rab11a, and expression of GTPase-defective Rab4aS22N and Rab11aS25N inhibited resensitization. After sustained stimulation (10(-7) M CGRP, >2 h), CLR/RAMP1 trafficked to lysosomes. RAMP1 was degraded approximately 4-fold more rapidly than CLR (RAMP1, 45% degradation, 5 h; CLR, 54% degradation, 16 h), determined by Western blotting. Inhibitors of lysosomal, but not proteasomal, proteases prevented degradation. Sustained stimulation did not induce detectable mono- or polyubiquitination of CLR or RAMP1, determined by immunoprecipitation and Western blotting. Moreover, a RAMP1 mutant lacking the only intracellular lysine (RAMP1K142R) internalized and was degraded normally. Thus, after transient stimulation with CGRP, CLR and RAMP1 traffic from endosomes to the plasma membrane, which mediates resensitization. After sustained stimulation, CLR and RAMP1 traffic from endosomes to lysosomes by ubiquitin-independent mechanisms, where they are degraded at different rates.

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    • "Thus, ECE-1 is expressed in RMA- SMCs, which also express mRNA transcripts for CLR and RAMP1. Therefore, if CGRP internalizes to endosomes with CLR@BULLETRAMP1, as observed in transfected HEK cells (Cottrell et al., 2007; Padilla et al., 2007), ECE-1 would be appropriately localized to degrade CGRP. In support of this finding, ECE-1-immunoreactivity has been detected in many types of human SMCs (Barnes and Turner, 1999; Granchi et al., 2002; Jackson et al., 2006). "
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    ABSTRACT: Background and Purpose: Calcitonin gene-related peptide (CGRP) is a potent vasodilator, implicated in the pathogenesis of migraine. CGRP activates a receptor complex comprising, calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1). In vitro studies indicate recycling of CLR•RAMP1 is regulated by degradation of CGRP in early endosomes by endothelin-converting enzyme-1 (ECE-1). However, it is not known if ECE-1 regulates the resensitization of CGRP-induced responses in functional arterial tissue. Experimental Approach: CLR, ECE-1a-d and RAMP1 expression in rat mesenteric artery smooth muscle cells (RMA-SMCs) and mesenteric arteries was analyzed by RT-PCR and by immunofluorescence and confocal microscopy. CGRP-induced signaling in cells was examined by measuring cAMP production and ERK activation. CGRP-induced relaxation of arteries was measured by isometric wire myography. ECE-1 was inhibited using the specific inhibitor, SM-19712. Key Results: RMA-SMCs and arteries contained mRNA for CLR, ECE-1a-d and RAMP1. ECE-1 was present in early endosomes of RMA-SMCs and in the smooth muscle layer of arteries. CGRP induced endothelium-independent relaxation of arteries. ECE-1 inhibition had no effect on initial CGRP-induced responses but reduced cAMP generation in RMA-SMCs and vasodilation in mesenteric arteries responses to subsequent CGRP challenges. Conclusions and Implications: ECE-1 regulates the resensitization of responses to CGRP in RMA-SMCs and mesenteric arteries. CGRP-induced relaxation does not involve endothelium-derived pathways. This is the first report of ECE-1 regulating CGRP responses in SMCs and arteries. ECE-1 inhibitors may attenuate an important vasodilatory pathway, implicated in primary headaches and may represent a new therapeutic approach for the treatment of migraine. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.
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    • "To exclude this possibility we examined the effects of bafilomycin A1, an inhibitor of vacuolar-type H+-ATPases, on CXCR7 recycling. It has previously been shown that endosomal acidification promotes dissociation of ligand from GPCRs, and that inhibitors of such acidification prevent receptor recycling and resensitization [33], [34]. As shown in Fig. 3D, incubation of CXCR7 expressing cells with 1 µM bafilomycin A1 did not affect CXCR7 internalization after 45 min of CXCL12 stimulation. "
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    • "The results indicate that ubiquitination of the hKOR is involved in targeting the receptor from endosomes to lysosomes. These results are consistent with those on several 7TMRs, including β 2 -AR (Shenoy et al., 2001), chemokine CXCR4 (Marchese and Benovic, 2001; Marchese et al., 2003), V2 vasopressin (Martin et al., 2003), NK-1 (Cottrell et al., 2006) and PAR2 (Jacob et al., 2005), but not those on the δ opioid receptor (Tanowitz and von Zastrow, 2002) and the complex of calcitonin receptor-like receptor and receptor activity-modifying protein 1 (Cottrell et al., 2007). Sorting of CXCR4 from endosomes to lysosomes requires Hrs and Vps4, an AAA ATPase; the latter is involved in regulating the ubiquitination status of both CXCR4 and Hrs (Marchese et al., 2003). "
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