Functional Complementation between a Novel Mammalian Polygenic Transport Complex and an Evolutionarily Ancient Organic Solute Transporter, OSTα-OSTβ

Abstract

These studies identify an organic solute transporter (OST) that is generated when two novel gene products are co-expressed, namely human OST␣ and OST␤ or mouse OST␣ and OST␤. The results also demonstrate that the mammalian proteins are functionally complemented by evolutionarily divergent Ost␣-Ost␤ proteins recently identified in the little skate, Raja erinacea, even though the latter exhibit only 25-41% predicted amino acid identity with the mammalian proteins. Human, mouse, and skate OST␣ proteins are predicted to contain seven transmembrane helices, whereas the OST␤ sequences are predicted to have a single transmembrane helix. Human OST␣-OST␤ and mouse Ost␣-Ost␤ cDNAs were cloned from liver mRNA, sequenced, expressed in Xenopus laevis oocytes, and tested for their ability to functionally complement the corresponding skate proteins by measuring transport of [ 3 H]estrone 3-sulfate. None of the proteins elicited a transport signal when expressed individually in oocytes; however, all nine OST␣-OST␤ combinations (i.e. OST␣-OST␤ pairs from human, mouse, or skate) generated robust estrone 3-sulfate transport activity. Transport was sodium-independent, saturable, and inhibited by other steroids and anionic drugs. Human and mouse OST␣-OST␤ also were able to mediate transport of taurocholate, digoxin, and prostaglandin E 2 but not of estradiol 17␤-D-glucuronide or p-aminohippurate. OST␣ and OST␤ were able to reach the oocyte plasma membrane when expressed either individually or in pairs, indicating that co-expression is not required for proper membrane targeting. Interestingly, OST␣ and OST␤ mRNAs were highly expressed and widely distributed in human tissues, with the highest levels occurring in the testis, colon, liver, small intestine, kidney, ovary, and adrenal gland.

OSTα-OSTβ: A major basolateral bile acid and steroid transporter in human intestinal, renal, and biliary epithelia

“…Note that although the human liver had relatively abundant OST␣ mRNA and a moderate amount of OST␤ mRNA, the mouse liver had very low levels of these transcripts. These human tissue Northern blot results generally confirm previous quantitative PCR analysis of human OST␣ and OST␤ 9 (Fig. 2C), with the only exceptions being the testes and colon, which gave weak signals on Northern blot analysis (Fig.…”

“…Both transcripts were relatively abundant in tissues that also express the apical sodium-dependent bile acid uptake transporter ASBT (SLC10A2), namely the human small intestine, kidney, and liver (Fig. 1), although they were also expressed at lower levels in many other tissues (data not shown; Seward et al 9 ). Note that although the human liver had relatively abundant OST␣ mRNA and a moderate amount of OST␤ mRNA, the mouse liver had very low levels of these transcripts.…”

“…14 Approximately 20 hours later, the cell monolayers were washed and incubated at 37°C for 10 to 60 minutes with Hank's balanced salt solution plus 10 mol/L of various radiolabeled bile acids added to the 15 and employed previously in our laboratory. [16][17][18] Oocytes were injected with 50 nL of human OST␣ and human OST␤ complementary RNA (cRNA) solutions (0.5-1 ng/oocyte), 9 or with water for controls. For uptake measurements, 6 to 8 oocytes were placed in a borosilicate glass culture tube per condition and incubated at 25°C in a modified Barth's solution containing various radiolabeled substrates.…”

“…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.…”

“…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.…”