👤 Michael R Dores

The presence of Mrap1 and Mrap2 orthologs in the genome of the elephant shark (es), a cartilaginous fish, presented an opportunity to evaluate the potential interactions between these accessory proteins and melanocortin receptors of a cartilaginous fish. RT-PCR analysis indicated that Mrap1 mRNA was present in interrenal, brain, and pituitary tissue with mRNA for Mc2R, Mc3R, Mc4R, and Mc5r. Co-expression of esMrap1 cDNA with esMc2r cDNA or esMc5r cDNA in CHO cells increased sensitivity to stimulation with ACTH(1-24) 10 fold and 100 fold, respectfully, but had no effect on sensitivity to stimulation with DesAc-αMSH [i.e., ACTH(1-13)NH Show less

The sorting of G protein-coupled receptors (GPCRs) to lysosomes is critical for proper signaling and cellular responses. We previously showed that the adaptor protein ALIX regulates lysosomal degradation of protease-activated receptor-1 (PAR1), a GPCR for thrombin, independent of ubiquitin-binding ESCRTs and receptor ubiquitination. However, the mechanisms that regulate ALIX function during PAR1 lysosomal sorting are not known. Here we show that the mammalian α-arrestin arrestin domain-containing protein-3 (ARRDC3) regulates ALIX function in GPCR sorting via ubiquitination. ARRDC3 colocalizes with ALIX and is required for PAR1 sorting at late endosomes and degradation. Depletion of ARRDC3 by small interfering RNA disrupts ALIX interaction with activated PAR1 and the CHMP4B ESCRT-III subunit, suggesting that ARRDC3 regulates ALIX activity. We found that ARRDC3 is required for ALIX ubiquitination induced by activation of PAR1. A screen of nine mammalian NEDD4-family E3 ubiquitin ligases revealed a critical role for WWP2. WWP2 interacts with ARRDC3 and not ALIX. Depletion of WWP2 inhibited ALIX ubiquitination and blocked ALIX interaction with activated PAR1 and CHMP4B. These findings demonstrate a new role for the α-arrestin ARRDC3 and the E3 ubiquitin ligase WWP2 in regulation of ALIX ubiquitination and lysosomal sorting of GPCRs. Show less