SITE-DIRECTED MUTAGENESIS OF P-2U PURINOCEPTORS - POSITIVELY CHARGED AMINO-ACIDS IN TRANSMEMBRANE HELIX-6 AND HELIX-7 AFFECT AGONIST POTENCY AND SPECIFICITY

Two subtypes of G protein coupled receptors for nucleotides (P-2U and P-2Y purinoreceptors) contain several conserved positively charged ami no acids in the third, sixth, and seventh putative transmembrane helic es (TMHs). Since the fully ionized form of nucleotides has been shown to be an activating ligand for both P-2U and P-2Y purinoceptors (P(2U) R and P(2Y)R), we postulated that some of these positively charged ami no acids are involved in binding of the negatively charged phosphate g roups of nucleotides. To investigate the role of the conserved positiv ely charged amino acids in purinoceptor function, a series of mutant P (2U)R cDNAs were constructed so that lysine 107 and arginine 110 in TM H 3, histidine 262 and arginine 265 in TMH 6, and arginine 292 in TMH 7 were changed to the neutral amino acid leucine or isoleucine, The mu tated P(2U)R cDNAs were stably expressed in 1321N1 astrocytoma cells a nd receptor activity was monitored by quantitating changes in the conc entration of intracellular Ca2+ upon stimulation with full (ATP, UTP) or partial (ADP, UDP) P(2U)R agonists, Neutralization of His(262), Arg (265), or Arg(292) caused a 100-850-fold decrease in the potency of AT P and UTP relative to the unmutated P(2U)R and rendered ADP and UDP in effective, In contrast, neutralization of Lys(107) or Arg(110) did not alter the agonist potency or specificity of the P(2U)R. Neutralizatio n of Lys(289) in the P(2U)R, which is expressed as a glutamine residue in the P-2Y subtype, did not alter receptor activity; however, a cons ersubtype, did not alter receptor activity; however, a conservative ch ange from lysine to arginine at this position altered the rank order o f agonist potency so that ADP and UDP were approximately 100-fold more potent than ATP and UTP, A three-dimensional model of the P(2U)R indi cates the feasibility of His(262), Arg(265), and Arg(292) interactions with the phosphate groups of nucleotides.