Role of conserved transmembrane cationic amino acids in the prostaglandin transporter PGT.
The prostaglandin transporter "PGT" interacts electrostatically with its anionic substrate, based on inhibition by the disulfonic stilbenes [Chan, B. S. (1998) J. Biol. Chem. 273, 6689-6697], inhibition by the thiol-reactive anion sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES) [Chan, B. S. (1999) J. Biol. Chem. 274, 25564-25570], and the requirement for a negatively charged 1-position carboxyl on the substrate [Itoh, S. (1996) Mol. Pharm. 50, 736-742]. Here we found that modification of positively charged residues on wild-type PGT by arginine- and lysine-specific reagents significantly inhibited transport. We previously found that the binding site of PGT is formed, at least in part, by its membrane-spanning segments [Chan, B. S. (1999) J. Biol. Chem. 274, 25564-25570]. Three charged residues within predicted transmembrane spans (E78, R560, and K613) are conserved in PGT and in related transporters. Substitution of the anionic residue E78 (E78D and E78C) produced an essentially functional transporter, whereas substitution of the cationic residues with neutral residues (R560N and K613Q) resulted in poorly functional transporters. Immunoblotting revealed similar expression levels of wild-type and mutant transporters, and immunostaining indicated correct targeting. Conservative charge substitutions (R560K, K613R, and K613H) resulted in generally functional transporters. In contrast, R560N was nonfunctional, whereas the substrate affinity of K613G decreased greater than 50-fold. Conservative substitutions retaining the charge at position 613 (K613R and K613H) restored the substrate affinity, suggesting a direct role of K613 in substrate binding. Double-neutral mutants E78G/R560C and E78G/K613C were inactive, indicating that these residues are not simply charge-paired. Our results suggest that an arginine at position 560 is critical for maximal substrate translocation, and that a positively charged side chain at position 613 contributes to electrostatic binding of the anionic substrate.
Renal Division, Department of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Brenda S
BS
Chan
Yi
Y
Bao
Victor L
VL
Schuster
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000502:physiology*; D000818:Animals; D017920:Antiporters / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D001483:Base Sequence; D017124:Conserved Sequence; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D007202:Indicators and Reagents / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D027361:Organic Anion Transporters; D051381:Rats; D017386:Sequence Homology, Amino Acid
D000596:Amino Acids; D017920:Antiporters; D017931:DNA Primers; D004268:DNA-Binding Proteins; D007202:Indicators and Reagents; D027361:Organic Anion Transporters; C503526:SLCO2A1 protein, human; C503528:Slco2a1 protein, rat