HUMAN P2Y(1) RECEPTOR - MOLECULAR MODELING AND SITE-DIRECTED MUTAGENESIS AS TOOLS TO IDENTIFY AGONIST AND ANTAGONIST RECOGNITION SITES

The molecular basis for recognition by human P2Y(1) receptors of the n ovel, competitive antagonist 2'-deoxy-N-6-methyladenosine 3',5'-bispho sphate (MRS 2179) was probed using site-directed mutagenesis and molec ular modeling. The potency of this antagonist was measured in mutant r eceptors in which key residues in the transmembrane helical domains (T Ms) 3, 5, 6, and 7 were replaced by Ala or other amino acids. The capa city of MRS 2179 to block stimulation of phospholipase C promoted by 2 -methylthioadenosine 5'-diphosphate (2-MeSADP) was lost in P2Y(1) rece ptors having F226A, K280A, or Q307A mutations, indicating that these r esidues are critical for the binding of the antagonist molecule. Mutat ion of the residues His132, Thr222, and Tyr136 had an intermediate eff ect on the capacity of MRS 2179 to block the P2Y(1) receptor. These po sitions therefore appear to have a modulatory role in recognition of t his antagonist. F131A, H277A, T221A, R310K, or S317A mutant receptors exhibited an apparent affinity for PARS 2179 that was similar to that observed with the wild-type receptor. Thus, Phe131, Thr221, His277, an d Ser317 are not essential for antagonist recognition. A computer-gene rated model of the human P2Y(1) receptor was built and analyzed to hel p interpret these results. The model was derived through primary seque nce comparison, secondary structure prediction, and three-dimensional homology building, using rhodopsin as a template, and was consistent w ith data obtained from mutagenesis studies. We have introduced a ''cro ss-docking'' procedure to obtain energetically refined 3D structures o f the Ligand-receptor complexes. Cross-docking simulates the reorganiz ation of the native receptor structure induced by a ligand. A putative nucleotide binding site was localized and used to predict which resid ues are likely to be in proximity to agonists and antagonists. Accordi ng to our model TM6 and TM7 are close to the adenine ring, TM3 and TM6 are close to the ribose moiety, and TM3, TM6, and TM7 are near the tr iphosphate chain.