Erb L, Garrad R, Wang Y, Quinn T, Turner J T, Weisman G A
Department of Biochemistry, University of Missouri, Columbia 65212.
J Biol Chem. 1995 Mar 3;270(9):4185-8. doi: 10.1074/jbc.270.9.4185.
Two subtypes of G protein-coupled receptors for nucleotides (P2U and P2Y purinoreceptors) contain several conserved positively charged amino acids in the third, sixth, and seventh putative transmembrane helices (TMHs). Since the fully ionized form of nucleotides has been shown to be an activating ligand for both P2U and P2Y purinoceptors (P2UR and P2YR), we postulated that some of these positively charged amino acids are involved in binding of the negatively charged phosphate groups of nucleotides. To investigate the role of the conserved positively charged amino acids in purinoceptor function, a series of mutant P2UR cDNAs were constructed so that lysine 107 and arginine 110 in TMH 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 mutated P2UR cDNAs were stably expressed in 1321N1 astrocytoma cells and receptor activity was monitored by quantitating changes in the concentration of intracellular Ca2+ upon stimulation with full (ATP, UTP) or partial (ADP, UDP) P2UR agonists. Neutralization of His262, Arg265, or Arg292 caused a 100-850-fold decrease in the potency of ATP and UTP relative to the unmutated P2UR and rendered ADP and UDP ineffective. In contrast, neutralization of Lys107 or Arg110 did not alter the agonist potency or specificity of the P2UR. Neutralization of Lys289 in the P2UR, which is expressed as a glutamine residue in the P2Y subtype, did not alter receptor activity; however, a conservative change from lysine to arginine at this position altered the rank order of agonist potency so that ADP and UDP were approximately 100-fold more potent than ATP and UTP. A three-dimensional model of the P2UR indicates the feasibility of His262, Arg265, and Arg292 interactions with the phosphate groups of nucleotides.
核苷酸的G蛋白偶联受体（P2U和P2Y嘌呤受体）的两个亚型在假定的第三、第六和第七个跨膜螺旋（TMH）中含有几个保守的带正电荷的氨基酸。由于已证明核苷酸的完全电离形式是P2U和P2Y嘌呤受体（P2UR和P2YR）的激活配体，我们推测这些带正电荷的氨基酸中的一些参与了核苷酸带负电荷的磷酸基团的结合。为了研究保守的带正电荷的氨基酸在嘌呤受体功能中的作用，构建了一系列突变型P2UR cDNA，使TMH 3中的赖氨酸107和精氨酸110、TMH 6中的组氨酸262和精氨酸265以及TMH 7中的精氨酸292变为中性氨基酸亮氨酸或异亮氨酸。突变型P2UR cDNA在1321N1星形细胞瘤细胞中稳定表达，通过定量完全（ATP、UTP）或部分（ADP、UDP）P2UR激动剂刺激后细胞内Ca2+浓度的变化来监测受体活性。His262、Arg265或Arg292的中和导致ATP和UTP的效力相对于未突变的P2UR降低100 - 850倍，并使ADP和UDP无效。相比之下，Lys107或Arg110的中和并未改变P2UR的激动剂效力或特异性。P2UR中赖氨酸289的中和（在P2Y亚型中以谷氨酰胺残基形式表达）并未改变受体活性；然而，该位置从赖氨酸到精氨酸的保守变化改变了激动剂效力的顺序，使得ADP和UDP的效力比ATP和UTP高约100倍。P2UR的三维模型表明His262、Arg265和Arg292与核苷酸磷酸基团相互作用的可行性。
