Expression cloning of two genes that together mediate organic solute and steroid transport in the liver of a marine vertebrate

Abstract

Uptake of organic solutes and xenobiotics by mammalian cells is mediated by ATP-independent transporters, and four families of transporters have now been identified. To search for novel organic solute transporters, a liver cDNA library from an evolutionarily primitive marine vertebrate, the little skate Raja erinacea, was screened for taurocholate transport activity by using Xenopus laevis oocytes. In contrast to the organic anion transporters identified to date, a transport activity was identified in this library that required the coexpression of two distinct gene products, termed organic solute transporter alpha and beta (Ostalpha, Ostbeta). Ostalpha cDNA encodes for a protein of 352 aa and seven putative transmembrane (TM) domains. Ostbeta contains 182 aa and has at least one and perhaps two TM domains. There is no significant sequence identity between Ostalpha and Ostbeta, and only low identity with sequences in the databases; however, Ostalpha bears a resemblance to some G protein-coupled receptors, and Ostbeta exhibits 22% amino acid identity with the C-terminal TM and intracellular domains of protocadherin-gamma, a cell surface glycoprotein. Xenopus oocytes injected with the cRNA for both Ostalpha and Ostbeta, but not each separately, were able to take up taurocholate, estrone sulfate, digoxin, and prostaglandin E(2), but not p-aminohippurate or S-dinitrophenyl glutathione. Transport was sodium-independent, saturable, and inhibited by organic anions and steroids, including the major skate bile salt, scymnol sulfate. These results identify an organic anion transporter composed of a putative seven-helix TM protein and an ancillary membrane polypeptide.

Figure 1

Screening of skate liver mRNA for [³H]taurocholate-transporting activity in Xenopus oocytes. (A) Taurocholate uptake in oocytes injected with either water (control, C), total skate liver mRNA (mRNA, 50 ng), or size-fractionated mRNA (F1–F6, 50 ng). Total skate liver mRNA was separated on a linear 6–26% (wt/wt) sucrose gradient, and oocytes were injected with mRNA collected from fractions 1–6 (F1, 3–4 kb; F2, 2.5–3.5 kb; F3, 2–3 kb; F4, 1.2–2.3 kb; F5, 0.8–1.8 kb; F6, 0.6–1.5 kb). After 3 days in culture, uptake of 1 μM [³H]taurocholate was measured at 25°C for 2 h and is reported as fmol/oocyte per 2 h. Values are means ± SE (n = 8). (B) Taurocholate uptake in oocytes injected with either water, a mixture of cRNA from 13 separate clones (M, 5 ng), or cRNA mixtures of 12 clones of which one clone was sequentially removed (M-1 to M-13, 5 ng each). After 3 days in culture, uptake of 20 μM [³H]taurocholate was measured at 25°C for 1 h and is reported as pmol/oocyte per 1 h. Values are means ± SE (n = 8). (C) Effect of various cRNA ratios of clone 4 (Ostα) and clone 12 (Ostβ) on [³H]taurocholate uptake in oocytes. Uptake of 20 μM [³H]taurocholate was measured at 25°C for 30 min, at 3 days after injection of cRNA, and is reported as pmol/oocyte per 30 min. Values are means ± SE (n = 4).

Figure 2

Nucleotide and deduced amino acid sequence for Ostα. Predicted TM domains are underlined and numbered 1–7, and the single putative N-linked glycosylation site (Asn-22) is indicated by *. A 6-nt possible polyadenylation signal is also underlined, and the first in-frame termination codon is noted by a period. This sequence has been submitted to GenBank (accession no. AY027664).

Figure 3

Nucleotide and deduced amino acid sequence for Ostβ. The two predicted TM domains are underlined and numbered. There are a total of four putative N-linked glycosylation sites (Asn-42, -48, -91, and -106); however, because two each are distributed on either side of one TM domain, only two can be extracellular. Asn-42 and -48 are predicted to be in an extracellular loop, and these are indicated by *. The 6-nt polyadenylation signal (AATAAA) also is underlined, and the first in-frame termination codon is noted by a period. This sequence has been submitted to GenBank (accession no. AY027665).

Figure 4

Northern blot and RT-PCR analysis of skate tissue RNA. (A and C) Five micrograms of mRNA from skate liver (L), kidney (K), intestine (I), pancreas (P), brain (B), and heart (H) were separated by 1.2% denaturing agarose gel electrophoresis, transferred to a nylon membrane, and UV-crosslinked. The blot was hybridized with ³²P-labeled full-length DNA fragments of Ostα (A) and Ostβ (C) under high stringency conditions. (B and D) RT-PCR was conducted on the same tissues using 0.5 μg total RNA as a template. The predicted 547-bp PCR product for Ostα was observed in liver and to a lesser extent in kidney. The predicted PCR fragment for Ostβ was detected in liver, kidney, intestine, and heart.

Figure 5

Substrate selectivity of Ostα/Ostβ. Oocytes were injected with either water, or 1 ng Ostα cRNA plus 1 ng of Ostβ cRNA, in 50 nl of water per oocyte. After 3 days in culture, the uptake of radiolabeled compounds ([³H]taurocholate, 20 μM; [³H]estrone sulfate, 50 nM; [³H]digoxin, 0.5 μM; [³H]prostaglandin E₂ (PGE₂), 5 nM; [³H]p-aminohippuric acid (PAH), 1 μM; or [³H]S-2,4-dinitrophenyl glutathione (DNP-SG), 0.1 μM) was measured at 25°C for 1 h. Uptake values are reported as pmol/oocyte per h for taurocholate and as fmol/oocyte per h for the other substrates. Values are means ± SE (n = 3–4).