Mapping the substrate binding site of the prostaglandin transporter PGT by cysteine scanning mutagenesis

Brenda S. Chan, Joe A. Satriano, Victor L. Schuster

Research output: Contribution to journalArticle

22 Scopus citations

Abstract

We have identified a cDNA, PGT, that encodes a widely expressed transporter for prostaglandin (PG) E2, PGF(2α), PGD2, 8-iso-PGF2α, and thromboxane B2. To begin to understand the molecular mechanisms of transporter function, we have initiated a structure-function analysis of PGT to identify its substrate-binding region. We have found that by introducing the small, water-soluble, thiol-reactive anion Na(2- sulfonatoethyl)methanethiosulfonate (MTSES) into the substrate pathway, we were able to cause inhibition of transport that could be reversed with dithiothreitol. Importantly, co-incubation with PGE2 protected PGT from this inhibition, suggesting that MTSES gains access to the aqueous pore pathway of PGT to form a mixed disulfide near the substrate-binding site. To identify the susceptible cysteine, we mutated, one at a time, all six of the putative transmembrane cysteines to serine. Only the mutation of Cys530 to serine within putative transmembrane 10 became resistant to inhibition by MTSES. Thus, Cys-530 is the substrate-protectable, MTSES-inhibitable residue. To identify other residues that may be lining the substrate-binding site, we initiated cysteine-scanning mutagenesis of transmembrane 10 using Cys-530 as an entry point. On a C530S, MTSES-resistant background, residues in the N- and C-terminal directions were individually mutated to cysteine (Ala-513 to His-536), one at a time, and then analyzed for MTSES inhibition. Of the 24 cysteine-substituted mutants generated, 6 were MTSES-sensitive and, among these, 4 were substrate-protectable. The pattern of sensitivity to MTSES places these residues on the same face of an α-helix. The results of cysteine-scanning mutagenesis and molecular modeling of putative transmembrane 10 indicate that the substrate binding of PGT is formed among its membrane-spanning segments, with 4 residues along the cytoplasmic end of helix 10 contributing to one surface of the binding site.

Original languageEnglish (US)
Pages (from-to)25564-25570
Number of pages7
JournalJournal of Biological Chemistry
Volume274
Issue number36
DOIs
StatePublished - Sep 3 1999

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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