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J. Biol. Chem., Vol. 282, Issue 16, 12260-12271, April 20, 2007

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

Graeme S. Cottrell, Benjamin Padilla, Stella Pikios, Dirk Roosterman, Martin Steinhoff, Eileen F. Grady, and Nigel W. Bunnett¹

From the Departments of Surgery and Physiology, University of California, San Francisco, California 94143-0660 and the Department of Dermatology, Interdisziplinäres Zentrum für Klinische Forschung Münster, and Ludwig Boltzmann Institute for Cell and Immunobiology of the Skin, University of Münster, Von-Esmarch-Strasse 58, 48149 Münster, Germany

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 [Ca²⁺]_i. Cycloheximide did not affect resensitization, but bafilomycin A₁, 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 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.

Received for publication, July 3, 2006 , and in revised form, January 9, 2007.

^* 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.

¹ To whom correspondence should be addressed: University of California San Francisco, 521 Parnassus Ave., San Francisco, CA 94143-0660. Tel.: 415-476-0489; Fax: 415-476-0936; E-mail: nigel.bunnett{at}ucsf.edu.

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