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 ␣ and ␤ (Ost␣, Ost␤). Ost␣ cDNA encodes for a protein of 352 aa and seven putative transmembrane (TM) domains. Ost␤ contains 182 aa and has at least one and perhaps two TM domains. There is no significant sequence identity between Ost␣ and Ost␤, and only low identity with sequences in the databases; however, Ost␣ bears a resemblance to some G protein-coupled receptors, and Ost␤ exhibits 22% amino acid identity with the C-terminal TM and intracellular domains of protocadherin-␥, a cell surface glycoprotein. Xenopus oocytes injected with the cRNA for both Ost␣ and Ost␤, but not each separately, were able to take up taurocholate, estrone sulfate, digoxin, and prostaglandin E 2, but not p-aminohippurate or Sdinitrophenyl 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. E pithelial cells continuously extract large amounts of organic solutes, drugs, and other xenobiotics from circulating blood plasma. Some of the transporters responsible for this drug clearance recently have been characterized at the molecular level and include four families of ATP-independent transporters: the Na ϩ -coupled bile acid transporters (NTCPs), the Na ϩ -independent organic anion transporting polypeptides (OATPs), the organic anion transporters (OATs), and the organic cation transporters (OCTs) (1-5). The first member of each of these families was identified by expression cloning in Xenopus laevis oocytes, and additional members subsequently have been identified by homology screening. All of these transporters consist of single polypeptides, which, when expressed in heterologous systems, are able to mediate organic solute transport.Because of the large number of endogenous and exogenous compounds that must be transported by the liver, kidney, intestine, and other tissues, it is likely that other transporters and transporter families also exist, but have not yet been described (5). In an attempt to identify novel organic anion transport proteins we used a comparative approach, screening a liver cDNA library from an evolutionarily ancient vertebrate species, the little skate Raja erinacea. This elasmobranch is thought to have evolved 200 million years ago, yet displays many physiological features...

OSTα‐OSTβ

“…Substrates for this transporter include the bile acid taurocholate, other steroids (estrone 3-sulfate and digoxin), and prostaglandin E 2 . 8,9 The present study demonstrates that another steroid, dehydroepiandrosterone sulfate (DHEAS), is also a substrate. The recent work of Dawson and colleagues 7 indicates that Ost␣ and Ost␤ messenger RNA (mRNA) expression along the mouse gastrointestinal tract mirrors that of Asbt, and that both Ost␣ and Ost␤ proteins are localized to the basolateral surface of ileal enterocytes.…”

“…In the little skate (Leucoraja erinacea), the liver is also a major site of Ost␣ and Ost␤ expression. 8 Although the basis for the species differences in tissue expression is unknown, it is interesting to note that recent studies have identified comparable differences between humans and mice in the expression of a cholesterol transporter, the Niemann-Pick C1-like 1 (NPC1L1) gene. 20 NPC1L1 encodes for a transporter that mediates the intestinal uptake of cholesterol and plant sterols, and appears to be critical in cholesterol homeostasis.…”

“…However, a recent study demonstrated that ileal basolateral bile acid export may be mediated by the Ost␣-Ost␤ heteromeric transporter, 7 a transporter that was initially identified in the liver of the little skate (Leucoraja erinacea). 8,9 In contrast to all other organic anion transporters identified to date, transport activity requires the coexpression of two distinct gene products: a predicted 340 -amino acid, 7-transmembrane domain protein (OST␣) and a putative 128 -amino acid, single-transmembrane domain ancillary polypeptide (OST␤). However, the mechanism through which these two proteins interact to generate transport activity has not yet been identified.…”

The Heteromeric Organic Solute Transporter α-β, Ostα-Ostβ, Is an Ileal Basolateral Bile Acid Transporter

“…One of the transporter genes that was significantly induced in the colon tissue of Slc10a2 null mice was the mouse ortholog of the organic solute transporter (Ost) ␣ subunit. Wang et al (17) originally identified the Ost transporter complex (Ost␣-Ost␤) from the little skate (Leucoraja erinacea) using expression cloning in Xenopus oocytes (17), and this same laboratory subsequently cloned and expressed the human and mouse orthologs of the skate Ost␣-Ost␤ proteins (14). Remarkably, solute transport requires the expression of two different subunits, a 340-amino acid polytopic membrane protein (Ost␣) and a 128-amino acid single trans-membrane protein (Ost␤), and when co-expressed, these two proteins mediate the transport of bile acids and other sterols (14,17).…”

“…Wang et al (17) originally identified the Ost transporter complex (Ost␣-Ost␤) from the little skate (Leucoraja erinacea) using expression cloning in Xenopus oocytes (17), and this same laboratory subsequently cloned and expressed the human and mouse orthologs of the skate Ost␣-Ost␤ proteins (14). Remarkably, solute transport requires the expression of two different subunits, a 340-amino acid polytopic membrane protein (Ost␣) and a 128-amino acid single trans-membrane protein (Ost␤), and when co-expressed, these two proteins mediate the transport of bile acids and other sterols (14,17). Moreover, human OST␣ and OST␤ are expressed in relative abundance in tissues that also express the ASBT, namely the human small intestine, kidney, and liver (14), suggesting that OST␣-OST␤ may be a basolateral bile acid exporter.…”