X Li

Publications (2)9.68 Total impact

• Source

  Available from: Jonathan Javitch

  Article: A cysteine residue in the third membrane-spanning segment of the human D2 dopamine receptor is exposed in the binding-site crevice.

  J A Javitch, X Li, J Kaback, A Karlin
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  ABSTRACT: The binding site in G-protein-linked neurotransmitter receptors is formed among their membrane-spanning segments. Because the binding site is in the plane of the bilayer and is accessible to charged, water-soluble agonists, it must lie in a crevice open to the extracellular, aqueous medium. Information about the structure of these receptors can be obtained by identifying the residues in the membrane-spanning segments which face this water-filled crevice. Human D2 dopamine receptor was expressed in human embryonic kidney 293 cells. Small, charged, sulfhydryl-specific methanethiosulfonate (MTS) derivatives irreversibly inhibited the binding of the D2-specific antagonist [3H]YM-09151-2 to these cells. The highly polar MTS derivatives should react with cysteine sulfhydryl groups only at the water-accessible surface of the receptor, which includes the surface of the binding-site crevice. In contrast, these reagents will have little access to sulfhydryls facing the lipid bilayer or buried in the protein interior. Positively charged MTS reagents irreversibly inhibited binding several hundredfold faster than a negatively charged MTS reagent, consistent with the affinity of the binding site for positively charged dopamine agonists and antagonists. Furthermore, both agonists and antagonists of the D2 receptor protected against irreversible inhibition by the MTS reagents. To identify the susceptible cysteine, we mutated, one at a time, five transmembrane and two extracellular cysteine residues to serine. Only the mutation of Cys118 to serine decreased the susceptibility of antagonist binding to irreversible inhibition by the MTS reagents. Thus, Cys118, a residue in the middle of the third membrane-spanning segment, is exposed in the D2 receptor binding-site crevice.
  Proceedings of the National Academy of Sciences 11/1994; 91(22):10355-9. · 9.68 Impact Factor
• Article: Expression and characterization of human dopamine D2 receptor in baculovirus-infected insect cells.

  J A Javitch, J Kaback, X Li, A Karlin
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  ABSTRACT: Multiple types of dopamine D2-like receptors (D2, D3, D4) have been identified. Differences in pharmacology among these receptors may have profound clinical ramifications for the treatment of psychosis. Analysis of the structure and function of their binding sites requires a source of large amounts of receptor, uncontaminated by the other types of D2-like receptor. We engineered a recombinant baculovirus containing the human D2 receptor cDNA (DRD2) to express this receptor in insect cells. Spodoptera frugiperda cells (Sf9 and Sf21) and Trichoplusia ni cells (TN-5) were infected with the recombinant baculovirus. Binding of the D2 antagonist [3H]YM-09151-2 to membranes fractions of these cells peaked at a specific activity of 5-8 pmol/mg protein, approximately 40 times that of membranes from bovine striatum. The receptor expressed in Sf9 cells was similar to that of striatum in its affinities for D2 agonists and antagonists. Sodium ion stimulated [3H]YM-09151-2 binding to D2 receptor in infected Sf9 cell membranes. This effect was fit by an allosteric model which predicted the apparent affinity of [3H]YM-09151-2. The D2 receptor expressed in Sf9 and TN-5 cells was photolabeled with N-(p-azido-m-[125I]iodophenylethyl)spiperone. The specifically labeled component(s) ran as a broad band of apparent molecular weight between 54,000 and 60,000. Deglycosylation of the labeled component(s) reduced its molecular weight to 46,000.
  Journal of receptor research 03/1994; 14(2):99-117.