Techniques to study specific cell-surface receptor-mediated cellular vitamin A uptake

Abstract

STRA6 is a multitransmembrane domain protein that was recently identified as the cell-surface receptor for plasma retinol-binding protein (RBP), the vitamin A carrier protein in the blood. STRA6 binds to RBP with high affinity and mediates cellular uptake of vitamin A from RBP. It is not homologous to any known receptors, transporters, and channels, and it represents a new class of membrane transport protein. Consistent with the diverse physiological functions of vitamin A, STRA6 is widely expressed in diverse adult organs and throughout embryonic development. Mutations in human STRA6 that abolish its vitamin A uptake activity cause severe pathological phenotypes in many human organs including the eye, brain, lung, and heart. This chapter describes functional assays for STRA6 in live cells and on cellular membranes. These assays can be employed to study the mechanism of this new membrane transport mechanism and its roles in the physiology and pathology of many organs.

Figure 1

Transmembrane topology of STRA6, the high-affinity cell-surface receptor for RBP (50). Transmembrane domains are indicated with Roman numerals. Missense mutations in human STRA6 associated with severe birth defects are indicated (47). Crystal structure of holo-RBP is based on structure 1HBP in Protein Data Bank.

Figure 2

Monitoring of the full absorption spectrum of the entire HPLC run during the holo-RBP purification. The peaks for holo-RBP and apo-RBP are indicated.

Figure 3

Vitamin A uptake activity from holo-RBP for human STRA6 mutants associated with severe birth defects (47, 49). Locations of these mutations in the transmembrane topology model of STRA6 are shown in Figure 1. The activity of wild-type STRA6 is defined as 100%.

Figure 4

HPLC assay for retinyl ester uptake using transfected or untransfected COS-1 cells that were incubated with 20% normal human serum for 4 hrs. Retinyl ester was extracted and analyzed by HPLC. Mobile phase was changed linearly from 100% methanol to 100% ethylacetate. Retinylpalmitate peaks are indicated.

Figure 5

Quantitation of RBP binding activities of human STRA6 mutants associated with severe birth defects (47, 49). Locations of these mutations in the transmembrane topology model of STRA6 are shown in Figure 1. The activity of wild-type STRA6 is defined as 100%.