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Post-endocytic sorting of calcitonin receptor-like receptor and receptor activity-modifying protein 1

Cottrell, G. S., Padilla, B., Pikios, S., Roosterman, D., Steinhoff, M., Grady, E. F. and Bunnett, N. W. (2007) Post-endocytic sorting of calcitonin receptor-like receptor and receptor activity-modifying protein 1. The Journal of Biological Chemistry, 282 (16). pp. 12260-12271. ISSN 1083-351X

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To link to this item DOI: 10.1074/jbc.M606338200

Abstract/Summary

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.

Item Type:Article
Refereed:Yes
Divisions:Faculty of Life Sciences > School of Chemistry, Food and Pharmacy > School of Pharmacy > Division of Pharmacology
No Reading authors. Back catalogue items
ID Code:30271
Uncontrolled Keywords:Animals Calcitonin Receptor-Like Protein Calcium/metabolism Cell Line Cycloheximide/pharmacology *Endocytosis Endosomes/metabolism Humans Intracellular Signaling Peptides and Proteins/*metabolism Lysosomes/metabolism Macrolides/pharmacology Membrane Proteins/*metabolism Protein Transport Rabbits Rats Receptor Activity-Modifying Protein 1 Receptor Activity-Modifying Proteins Receptors, Calcitonin/*metabolism Transfection
Publisher:American Society for Biochemistry and Molecular Biology

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