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

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

doi:10.1074/jbc.m606338200

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.

Highlights

Specific alternate splicing of transcripts from the calcitonin gene [1]
After 10 min with calcitonin gene-related peptide (CGRP), calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1) were depleted from the plasma membrane and were colocalized with EEA1 in early endosomes (Fig. 1, A and B, white arrows)
At earlier times (e.g. 60 min), CLR and LAMP1 were rarely detected in lysosomes. These results show that activated CLR and RAMP1 traffic from the plasma membrane to early endosomes and lysosomes, which is consistent with other reports of CLR and RAMP1 trafficking [11]

Summary

Introduction

Specific alternate splicing of transcripts from the calcitonin gene [1]. CGRP is a potent vasodilator and mediator of neurogenic inflammation and pain transmission [2, 3]. For neuropeptides, the CGRP receptor is a heterodimer composed of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1). Upon activation with CGRP, CLR, but not RAMP1, is phosphorylated and interacts with ␤-arrestins [12]. 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. Trafficking of CLR and RAMP1 oid receptor, neurokinin 3 receptor) form low affinity and transient interactions with ␤-arrestin and rapidly recycle [13, 14]. It is not known whether CLR and RAMP1 can recycle

Objectives

Results

Conclusion