Xenopus autosomal recessive hypercholesterolemia protein couples lipoprotein receptors with the AP-2 complex in oocytes and embryos and is required for vitellogenesis.
ARH is required for normal endocytosis of the low density lipoprotein (LDL) receptor in liver and mutations within this gene cause autosomal recessive hypercholesterolemia in humans. xARH is a localized maternal RNA in Xenopus with an unknown function in oogenesis and embryogenesis. Like ARH, xARH contains a highly conserved phosphotyrosine binding domain and a clathrin box. To address the function of xARH, we examined its expression pattern in development and used pull-down experiments to assess interactions between xARH, lipoprotein receptors and proteins in embryo extracts. xARH was detected concentrated at the cell periphery as well as in the perinuclear region of oocytes and embryos. In pull-down experiments, the xARH phosphotyrosine binding domain interacted with the LDL and vitellogenin receptors found in Xenopus oocytes and embryos. Mutations within the receptor internalization signal specifically abolished this interaction. The xARH C-terminal region pulled-down several proteins from embryo extracts including alpha- and beta-adaptins, subunits of the AP-2 endocytic complex. Mutations within either of the two Dvarphi(F/W) motifs found in xARH abolished binding to alpha- and beta-adaptins. Expression of a dominant negative mutant of xARH missing the clathrin box and one functional Dvarphi(F/W) motif severely inhibited endocytosis of vitellogenin in cultured oocytes. The data indicate that xARH acts as an adaptor protein linking LDL and vitellogenin receptors directly with the AP-2 complex. In oocytes, we propose that xARH mediates the uptake of lipoproteins from the blood for storage in endosomes and later use in the embryo. Our findings point to an evolutionarily conserved function for ARH in lipoprotein uptake.
Zhou
Yi
Y
Department of Cell Biology and Anatomy, University of Miami School of Medicine, Miami, Florida 33136, USA.
Zhang
Jian
J
King
Mary Lou
ML
D033962:Adaptor Protein Complex 2 / Q000378:metabolism*; D033965:Adaptor Protein Complex alpha Subunits / Q000737:chemistry / Q000378:metabolism; D033966:Adaptor Protein Complex beta Subunits / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004527:Egg Proteins / Q000378:metabolism; D004625:Embryo, Nonmammalian / Q000737:chemistry / Q000378:metabolism*; D005260:Female; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000737:chemistry / Q000378:metabolism*; D019000:Phosphotyrosine; D011956:Receptors, Cell Surface / Q000378:metabolism; D011973:Receptors, LDL / Q000378:metabolism; D018110:Receptors, Lipoprotein / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D014818:Vitellogenesis; D014981:Xenopus / Q000196:embryology; D029867:Xenopus Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*
D033962:Adaptor Protein Complex 2; D033965:Adaptor Protein Complex alpha Subunits; D033966:Adaptor Protein Complex beta Subunits; D004527:Egg Proteins; D011956:Receptors, Cell Surface; D011973:Receptors, LDL; D018110:Receptors, Lipoprotein; D011993:Recombinant Fusion Proteins; D029867:Xenopus Proteins; C480042:autosomal recessive hypercholesterolemia protein, Xenopus; C030474:vitellogenin receptor; D019000:Phosphotyrosine; D005982:Glutathione Transferase