The effect of missense mutations in the RhoGAP-homology domain on ocrl1 function.

Lowe syndrome is a rare X-linked disease characterized by congenital cataracts, defects in renal tubule cell function, and mental retardation. Mutations in the OCRL1 gene, which encodes ocrl1, a phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2)) 5-phosphatase, are the cause of Lowe syndrome. PtdIns(4,5)P(2), a substrate of ocrl1, is an important signaling molecule within the cell. OCRL1 is ubiquitously expressed and co-localizes with the trans-Golgi network (TGN) and endosomal proteins. The ocrl1 protein contains two recognizable domains, one a conserved Ptd(4,5)P(2) 5-phosphatase domain and the other with homology to Rho GTPase activating proteins (RhoGAPs). The objective of our study was to further characterize the ocrl1 RhoGAP-homology domain by analyzing the effect of two missense mutations in this domain, I751N and A780P, which were previously reported in Lowe syndrome patients. Both mutant proteins were expressed at levels similar to wild-type but their enzyme activity was reduced by 85-90%, indicating that the RhoGAP-homology domain is important for the enzymatic function of ocrl1. Study of a C-terminal region of wild-type ocrl1 containing this domain detected no GAP activity, eliminating the possibility of an effect by mutations in this domain on GTPase activation. Because members of the Arf family of small G-proteins are directly involved in (Ptd(4,5)P(2)) signaling and localize to the TGN like ocrl1, we analyzed by immunoprecipitation the interaction of ocrl1 with Arf1 and Arf6 via its RhoGAP-homology domain. Wild-type ocrl1, but not the I751N mutant protein, co-immunoprecipitated with these two Arf proteins. These results indicate that wild-type ocrl1 and Arf proteins can interact and that this interaction is disrupted by the mutation. It remains unknown whether a disrupted interaction between Arf and ocrl1 plays a role in the Lowe syndrome phenotype.