Rab GTPases, which cycle between GDP-bound inactive and GTP-bound active states, are central elements of the vesicular transport machinery. Importantly, Rab5 is a key element of early endocytosis and trafficking. Guanine nucleotide exchange factors (GEFs) control Rab activity and various proteins have already been identified as Rab5 GEFs.
These proteins all contain a highly conserved Vps9-domain that serves as GEF domain for Rab5 family GTPases, including Rab21, Rab22a and Rab31. A family of multifunctional Ras and Rab interactors (RINs) has been described that contain a N-terminal SH2 domain and RH, Vps9 and Ras-association domains in their C-terminal portion. At present, three RIN family members have been described and analyzed. They all serve as GEFs for Rab5 and at least one additional Rab5 family GTPase and seem to be involved in endocytosis and vesicular transport mechanisms.
Here we report the identification of a novel modulator of Rab5 activity, the Vps9 domain containing protein Rin-like (Rinl). Rinl shares high sequence homology and the overall domain structure with the RIN proteins but lacks the RA domain at its C-terminus. Noteworthy, Rinl has been isolated as new interaction partner of the muscle specific receptor tyrosine kinase MuSK and interacts with MuSK independent of its phosphorylation. Signal transduction events triggered by MuSK are essential for neuromuscular synapse (NMS) formation and maintenance.
Experimental evidence shows that Rinl is localized to NMSs but the highest expression levels have been found in thymus and spleen. Rinl preferentially binds to GDP-bound Rab5a and catalyzes the exchange of GDP for GTP. The same interaction of Rinl was also found for GDP-Rab22a, but strikingly Rinl has a much stronger GEF activity towards Rab22a than Rab5a. Besides a diffuse, cytoplasmic distribution Rinl displays a characteristic localization pattern in vesicles and actin-positive membrane ruffles, implicating Rinl in endocytotic processes involving Rab5a and Rab22a. The Rinl Vps9 domain but not the SH2 domain is required for catalytic activity and cytoskeleton-associated membrane localization. Most importantly, overexpression of Rinl increases fluid-phase uptake as well as epidermal growth factor receptor endocytosis.
Besides the biochemical characterization of Rinl a gene targeting approach in mice has been performed, aiming to elucidate Rinl function and expression in vivo. Rinl-/- mice are viable and fertile. NMSs appear to develop normally and do not show any gross alterations in size and shape.