Abstract
The Xenopus homologue of hARH (A&barbelow;utosomal R&barbelow;ecessive H&barbelow;ypercholesterolemia) was identified in a screen for maternal vegetally localized RNAs. xARH contains an N-terminal phosphotyrosine binding (PTB) domain 91% identical to that of human ARH. Unlike the human homologue, maternal xARH occurs as two transcripts that differ in 3' UTRs. The large transcript, xARH-alpha, localizes primarily to the vegetal cortex. The small transcript, xARH-beta, is present almost exclusively in the animal half. xARH is detected cortically in early stage oocytes. The protein is also found in the animal half of eggs and in the perinuclear region of blastomeres in embryos. Both xARH transcripts are expressed in the adult liver, but at levels lower than in oocytes. The PTB domain binds to the cytoplasmic tail of receptors bearing NPXY motifs including the LDL and vitellogenin receptors. Several proteins that associate with the C-terminal region of xARH-alpha were purified. One of them, p108, is alpha-adaptin as shown by mass spectrometry and western blot analysis. Alpha-adaptin is a subunit of the clathrin adaptor complex, AP-2, which mediates protein endocytosis from plasma membrane to endosome. The GST-xARH C-terminal fusion protein also pulled down other subunits of the AP-2 complex. The interaction between xARH C and the AP-2 complex requires D&phis;F/W motifs which bind to the appendage domains of alpha and beta adaptins. xARH also contains a conserved clathrin box, and thus may bind to clathrin. ARH homologues have been found in mosquito, ascidian, zebrafish, mouse and human. ARH appears to be an ancient gene involved in lipoprotein receptor internalization. My data are consistent with xARH acting as an adaptor protein linking receptor(s) and the endocytic machinery. In oocytes, xARH may mediate the uptake of LDL and vitellogenin from the blood, and is essential for oogenesis. The two xARH transcripts may encode proteins with slightly different functions, accounting for their different localization pattern.