Mapping the membrane topology and extracellular ligand binding domains of the retinol binding protein receptor.
STRA6 is a multitransmembrane domain protein not homologous to any other proteins with known function. It functions as the high-affinity receptor for plasma retinol binding protein (RBP) and mediates cellular uptake of vitamin A from the vitamin A-RBP complex. Consistent with the diverse roles of vitamin A and the wide tissue expression pattern of STRA6, mutations in STRA6 are associated with severe pathological phenotypes in humans. The structural basis for STRA6's biochemical function is unknown. Although computer programs predict 11 transmembrane domains for STRA6, its topology has never been studied experimentally. Elucidating the transmembrane topology of STRA6 is critical for understanding its structure and function. By inserting an epitope tag into all possible extracellular and intracellular domains of STRA6, we systematically analyzed the accessibility of each tag on the surface of live cells, the accessibility of each tag in permeabilized cells, and the effect of each tag on RBP binding and STRA6-mediated vitamin A uptake from the vitamin A-RBP complex. In addition, we used a new lysine accessibility technique combining cell-surface biotinylation and tandem-affinity purification to study a region of the protein not revealed by the epitope tagging method. These studies not only revealed STRA6's extracellular, transmembrane, and intracellular domains but also implicated extracellular regions of STRA6 in RBP binding.
Kawaguchi
Riki
R
Department of Physiology, Jules Stein Eye Institute, and Brain Research Institute, David Geffen School of Medicine at UCLA, Los Angeles, California 90095, USA.
Yu
Jiamei
J
Wiita
Patrick
P
Ter-Stepanian
Mariam
M
Sun
Hui
H
D000818:Animals; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002463:Cell Membrane Permeability; D002470:Cell Survival; D002522:Chlorocebus aethiops; D005110:Extracellular Space / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008954:Models, Biological; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D050800:STAT6 Transcription Factor / Q000235:genetics / Q000378:metabolism; D014801:Vitamin A / Q000378:metabolism
D008024:Ligands; D011956:Receptors, Cell Surface; D050800:STAT6 Transcription Factor; C027928:retinol binding protein receptor; D014801:Vitamin A