The Role of Ubiquitination and Hepatocyte Growth Factor-Regulated Tyrosine Kinase Substrate in the Degradation of the Adrenomedullin Type I Receptor

Introduction: Adrenomedullin (AM) receptors comprise the G protein-coupled receptor (GPCR), calcitonin receptor-like receptor (CLR) and a single transmembrane protein, receptor activity-modifying protein (RAMP). Three RAMP family members can heterodimerize with CLR to form functional receptors. CLR•RAMP1 is a high affinity calcitonin gene-related peptide (CGRP) receptor, whereas CLR•RAMP2 and CLR•RAMP3 are AM1 and AM2 receptors (1). CGRP receptors can recycle following transient stimulation (a process that requires endosomal cleavage of CGRP) or are degraded following sustained stimulation (a process that is independent of ubiquitination and dependent on the endosomal sorting protein, hepatocyte growth factor-regulated tyrosine kinase substrate (HRS)) (2,3,4). However, much less is known about the mechanisms controlling the post-endocytic sorting of CLR•RAMP2. Therefore, we determined the mechanisms that regulate the trafficking of CLR•RAMP2.

Methods: The trafficking of CLR•RAMP2 was studied in HEK cells. AM-induced ubiquitination of CLR•RAMP2 and CLRΔ9KR•RAMP2 (a lysine-less mutant) were examined using immunoprecipitation and western blotting. AM-induced trafficking of CLR•RAMP2 using immunofluorescence and confocal microscopy. AM-induced degradation of CLR•RAMP2 and CLRΔ9KR•RAMP2 was examined using a cell-surface biotinylation assay and western blotting.

Results: AM (100 nM, 60 min) promoted ubiquitination of CLR in HEK-CLR•RAMP2 cells (4.8±1.2 fold over basal) but not RAMP2 or CLRΔ9KR in HEK-CLRΔ9KR•RAMP2 cells. AM induced trafficking of CLR•RAMP2 and CLRΔ9KR•RAMP2 to endosomes (30 min) and lysosomes (4 h), indicating that ubiquitination does not regulate lysosomal targeting of AM1 receptors. Sustained (AM, 100 nM, 16 h) and transient (AM, 100nM, 30 min+16 h recovery) stimulation of CLR•RAMP2 and CLRΔ9KR•RAMP2 promoted degradation of receptor components to similar levels (sustained (CLR, 7.5±3.5%; CLRΔ9KR, 5.4±1.9%; RAMP2 (CLR) 21±5%; RAMP2 (CLRΔ9KR) 14±7.5%; transient (CLR, 73±3%; CLRΔ9KR, 62±8%; RAMP2 (CLR) 72±1%; RAMP2 (CLRΔ9KR) 60±3%, compared to untreated controls [100%]). Ubiquitination of GPCRs has been shown to regulate degradation kinetics (5); therefore, we examined degradation after a shorter exposure (4 h) to AM. No differences in degradation were observed (CLR, 72±1.1%; CLRΔ9KR 71±5.9%; RAMP2 (CLR) 73±8.9; RAMP2 (CLRΔ9KR) 49±9.3%, compared to untreated controls [100%]). Finally, we investigated the role of HRS in the degradation of AM1 receptors. Overexpression of HRS prevented AM-induced (100 nM, 4 h) degradation of CLR•RAMP2 but not CLRΔ9KR•RAMP2 (CLR, 102±7.9%; CLRΔ9KR 56±15%; RAMP2 (CLR) 85±11%; RAMP2 (CLRΔ9KR) 47±4.1%, compared to untreated controls [100%]). Results are expressed as mean±S.E.M. of n≥3 experiments and were compared by Student’s t test.

Conclusions: Our data shows that sustained and transient stimulation with AM promotes degradation of CLR•RAMP2 and CLRΔ9KR•RAMP2 and supports a role for ubiquitination and HRS in AM-induced degradation of CLR•RAMP2.

References:

(1) McLatchie, L. M. et al. (1998) Nature 393, 333-339.

(2) Cottrell, G. S. et al. (2007) J Biol Chem 282, 12260-12271.

(3) Padilla, B. E. et al. (2007) J Cell Biol 179, 981-997.

(4) Hasdemir, B. et al. (2007) J Biol Chem 282, 29646-29657.

(5) Hislop, J. N. et al. (2009) J Biol Chem 284, 19361-19370.