Extracellular ATP4- modulates organic anion transport by rat hepatocytes

Campbell, Celeste G.; Spray, David C.; Wolkoff, Allan W.

doi:10.1016/s0021-9258(18)82271-2

Cited by 24 publications

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“…In earlier studies, we demonstrated that hepatocyte uptake of the oatp1a1 substrate sulfobromophthalein (5) is down-regulated rapidly, specifically, and reversibly by extracellular ATP (6), an event that coincides with serine phosphorylation of oatp1a1 at a single tryptic phosphopeptide (7). The location of this peptide within the oatp1a1 sequence is unknown and is the subject of the present study.…”

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confidence: 67%

“…Expression of these proteins can be regulated at the transcriptional level, and a role for specific transcription factors and nuclear hormone receptors has been demonstrated (27)(28)(29). However, this is a relatively slow process and cannot explain the rapid modulation of transport function that has been described following exposure of rat hepatocytes to extracellular ATP or PKC activators, conditions that result in phosphorylation of oatp1a1 (6,7,11). Although previous studies showed that a single tryptic peptide was phosphorylated on a serine residue (7), there was no information as to where this peptide was located in the oatp1a1 sequence.…”

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confidence: 99%

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Rat Organic Anion Transporting Protein 1A1 (Oatp1a1): Purification and Phosphopeptide Assignment

et al. 2006

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Rat organic anion transporting protein 1a1 (oatp1a1), a hepatocyte basolateral plasma membrane protein, mediates transport of various amphipathic compounds. Our previous studies indicated that serine phosphorylation of a single tryptic peptide inhibits its transport activity without changing its cell surface content. The site of phosphorylation is unknown and was the subject of the present study. Following immunoaffinity chromatographic purification from rat liver, oatp1a1 was subjected to trypsin digestion and MALDI-TOF. Except for predicted N-glycosylated peptides, 97% of oatp1a1 tryptic peptides were observed. A single tryptic phosphopeptide was found in the C-terminus (aa 626-647), existing in unphosphorylated, singly, or doubly phosphorylated forms, and sensitive to alkaline phosphatase treatment. β-elimination reaction resulted in mass loss of 98 or 196 Da from this peptide, and subsequent Michael addition with cysteamine increased masses by the predicated 77 and 154 Da, indicating that oatp1a1 can be singly or doubly phosphorylated at serine or threonine residues in the C-terminal sequence SSATDHT (aa 634-640). Subsequent tandem MS/MS analysis revealed that phosphorylation at S634 accounted for all singly phosphorylated peptide, while phosphorylation at S634 and S635 accounted for all doubly phosphorylated peptide. These findings identify the site of oatp1a1 phosphorylation and demonstrate that it is an ordered process, in which phosphorylation at S634 precedes that at S635. The mechanism by which phosphorylation results in loss of transport activity in hepatocytes remains to be established. Whether phosphorylation near the C-terminus inhibits C-terminal oligomerization of oatp1a1, required for normal transport function, can be speculated upon, but is as yet unknown.The rat organic anion transporting protein 1a1 (oatp1a1) is expressed on the basolateral plasma membrane of hepatocytes, on the apical plasma membrane of the S3 segment of the proximal tubule, and on the apical plasma membrane of the choroid plexus epithelial cell (1;2). This protein, formerly known as oatp1, has recently been renamed oatp1a1 in a proposal for standardization of nomenclature (3). It, as well as other members of the oatp family, have been shown to transport a wide variety of amphipathic organic compounds (3;4) and are thought to be involved in a broad range of physiological, pathophysiological, and pharmacological processes (3;4). Although function of the oatp's has been studied extensively, their structure and regulation remain relatively unknown.In earlier studies, we demonstrated that hepatocyte uptake of the oatp1a1 substrate sulfobromophthalein (5) is down-regulated rapidly, specifically, and reversibly by extracellular ATP (6), an event that coincides with serine phosphorylation of oatp1a1 at a single tryptic † This work was supported by NIH grants DK23026 and CA101150. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript phosphopeptide(7). The location of this peptide within the oatp1a1 sequ...

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confidence: 67%