Linking GASP-1 dependent sorting to the ESCRT machinery

Seven transmembrane (7TM)/G protein coupled receptors (GPCRs) convey the majority of signal transduction across the cell membrane and are thus extensively regulated under normal conditions. One way in which their activity is controlled is by the mechanism of endocytosis, whereby agonist-activated receptors are rapidly and reversibly silenced through removal from the cell surface. Following endocytosis, individual receptors can be sorted differentially between recycling endosomes and lysosomes, which contributes to receptor regulation in two ways that have opposing effects on cell signaling. The sorting of receptors to the lysosomal pathway can be facilitated by the interaction with an array of proteins. The highly conserved ESCRT (endosomal sorting complex required for transport) machinery for example directs the transport of ubiquitinated cargo for degradation. Recently, the GPCR-associated sorting protein 1 (GASP-1) was identified, that specifically targets several 7TM/GPCRs to the lysosomal pathway by a yet unknown mechanism (Whistler et al., 2002). Intriguingly, ubiquitination of several mammalian 7TM/GPCRs does not appear to be a prerequisite for degradation, although the ESCRT machinery is required. Together these data suggest that the ESCRT machinery in some way can recognize and select non- ubiquitinated receptors and transport them to the lysosomes. Two recent key findings of the Whistler laboratory, (i) that GASP-1 itself is ubiquitinated and (ii) that GASP-1 interacts with TSG101, a subunit of the mammalian ESCRT-I complex, suggest a mechanism by which 7TM/GPCRs could be recognized and degraded by the ESCRT machinery. Based on these preliminary data, I aim to investigate whether GASP-1 might act as ubiquitinated `linker` between the ESCRT machinery and non- ubiquitinated receptors that are targeted for degradation. In this proposal I will identify interaction partners of GASP-1 within the ESCRT machinery and also map the sites on GASP-1 necessary for both interaction with ESCRT and its ubiquitination. Additionally, I will evaluate the functional consequences of disruption of these interactions by monitoring the trafficking of two 7TM/GPCRs that are sorted to the lysosomal pathway by GASP-1 - the cannabinoid 1 receptor (CB1R) and the dopamine 2 receptor (D2R). Recently, the relevance of GASP-1 dependent sorting of 7TM/GPCRs was also verified in vivo. In GASP-1 knock-out mice it was shown that the downregulation of CB1R is decreased, which led to a reduced development of tolerance after repeated dosage of WIN55,212-2 (Martini et al., 2010). Additionally, GASP-1 was also suggested to be implicated in the acute and chronic behavioural responses induced by cocaine (Boeuf et al., 2009). Hence, I will also translate my basic/mechanistic findings on receptor sorting into GASP-1 animal models. Summarized, these investigations will bring us one step closer in unravelling the complex sorting events that control the activity of 7TM/GPCRs. Furthermore, the evaluation of the functional consequences of receptor trafficking and their deregulation in vivo might lead to a better understanding of the relevance of these processes in different disease states.