Effect of two tyrosine mutations on the activity and regulation of the renal type II Na/P-i-cotransporter expressed in oocytes

The rat renal type II Na/Pi-cotransporter (NaPi2), which is regulated by me chanisms involving endocytosis and lysosomal degradation, contains two sequ ences that show high homology with two tyrosine (Y)-based consensus motifs previously reported to be involved in such intracellular trafficking: GY(40 2)FAM matching the consensus sequence GYXXZ, and Y509RWF matching the motif YXXO. Mutations of any of these two Y nearly abolished the NaPi2 mediated P-32(i)-uptake after cRNA-injection into oocytes. The mechanisms underlying these defects are however different. Mutation of the Y402 results in a lac k of glycosylation and reduced surface expression of the cotransporter, tha t are specific for the Y402 mutation since substitution of the neighboring F403 did not have any effect. The inhibitory effect of the Y509 mutation is related to a functional inactivation of the protein expressed in the plasm a membrane; mutation of the neighboring R510 also led to a decrease in the cotransporter activity. Pharmacological activation of the protein kinase C cascade by DOG induced the retrieval of both wild-type (WT) as well as Y509 cotransporters from the oocyte plasma membrane. These data suggest that th e Y402 is important for the surface expression whereas Y509 for the functio n of the type II Na/P-i-cotransporter expressed in oocytes. Y509 seems not to be involved in the membrane retrieval of the cotransporter.