ABSTRACT
The P2Y(12) receptor, a Gi protein-coupled receptor, plays a central role in platelet activation. In this study, we did a mutational analysis of residues possibly involved in the ligand interactions with the human P2Y(12) receptor. Mutant receptors were stably expressed in CHO-K1 cells with an HA-tag at the N-terminus. Expression of wild-type and mutant receptors was confirmed by detecting the HA-tag on the cell membrane. Residues in transmembrane helical domains (TMs) 3, 5, 6, and 7, which are homologous to residues important for P2Y(1) receptor activation and ligand recognition, were replaced by site-directed mutagenesis. ADP-induced inhibition of forskolin-stimulated cAMP levels in the presence or absence of antagonist AR-C69931MX were investigated for each of the mutant receptors. F104S and S288P significantly increased agonist-induced receptor function without affecting the antagonism by AR-C69931MX. Arg256 in TM6 and Arg 265 in extracellular loop 3 (EL3) are more important for antagonist recognition than effect on agonist-mediated receptor function. Compared to wild-type P2Y(12) receptor, mutations in Arg 256 or/and Arg 265 significantly increased the sensitivity to antagonist AR-C69931MX. Our study shows that the cytosolic side of TM3 and the exofacial side of TM5 are critical for P2Y(12) receptor function, which is different from P2Y(1). Arg 256 in TM6 and Arg265 in EL3 appear to play a role in antagonist recognition rather than effects on agonist-induced receptor function.
Subject(s)
Adenosine Diphosphate/pharmacology , Cyclic AMP/metabolism , Receptors, Purinergic P2Y12/metabolism , Adenosine Diphosphate/metabolism , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/metabolism , Adenosine Monophosphate/pharmacology , Animals , CHO Cells , Cricetinae , Cricetulus , DNA Mutational Analysis/methods , Humans , Ligands , Mutagenesis, Site-Directed , Protein Binding , Receptors, Purinergic P2Y12/drug effects , Receptors, Purinergic P2Y12/geneticsABSTRACT
The human prostaglandin EP3 receptor comprises eight isoforms that differ in carboxyl-tail. We show here that the isoforms are trafficked differently. When expressed in HEK293 cells, the isoforms located to the cell surface, although a fraction of some remained in the cell. Upon prostaglandin E(2) stimulation, EP3.I internalized almost completely, EP3.II, EP3.V, EP3.VI and EP3.f internalized to a lesser extent and EP3.III and EP3.IV did not internalize. Both EP3.I and EP3.f internalized with beta-arrestin and internalization were blocked by a dominant negative form of Eps15, a clathrin-associated protein. Although EP3.II internalized, beta-arrestin did not translocate with the receptor and internalization was not blocked by mutant Eps15. EP3.V and EP3.VI internalized to discrete areas of the cell with beta-arrestin.
Subject(s)
Dinoprostone/pharmacology , Receptors, Prostaglandin E/metabolism , Cell Line , DNA Primers , Female , Humans , Kidney , Mutagenesis, Site-Directed , Protein Isoforms/agonists , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Transport , Receptors, Prostaglandin E/agonists , Receptors, Prostaglandin E/genetics , Receptors, Prostaglandin E, EP2 Subtype , Receptors, Prostaglandin E, EP3 Subtype , Recombinant Proteins/metabolism , Restriction Mapping , Transfection , UterusABSTRACT
Phospholipase Cgamma2 (PLCgamma2) has been implicated in collagen-induced signal transduction in platelets and antigen-dependent signaling in B-lymphocytes. It has been suggested that tyrosine kinases activate PLCgamma2. We expressed the full-length cDNA for human PLCgamma2 in bacteria and purified the recombinant enzyme. The recombinant enzyme was Ca(2+)-dependent with optimal activity in the range of 1 to 10 microM Ca(2+). In vitro phosphorylation experiments with recombinant PLCgamma2 and recombinant Lck, Fyn, and Lyn tyrosine kinases showed that phosphorylation of PLCgamma2 led to activation of the recombinant enzyme. Using site-directed mutagenesis, we investigated the role of specific tyrosine residues in activation of PLCgamma2. A mutant form of PLCgamma2, in which all three tyrosines at positions 743, 753, and 759 in the SH2-SH3 linker region were replaced by phenylalanines, exhibited decreased Lck-induced phosphorylation and completely abolished the Lck-dependent activation of PLCgamma2. Individual mutations of these tyrosine residues demonstrated that tyrosines 753 and 759, but not 743, were responsible for Lck-induced activation of PLCgamma2. To confirm these results, we procured a phosphospecific antibody to a peptide containing phosphorylated tyrosines corresponding to residues 753 and 759. This antibody recognized phosphorylated wild-type PLCgamma2 on Western blots but did not interact with unphosphorylated PLCgamma2 or with PLCgamma2 containing mutated tyrosine residues at 753 and 759. Using this antibody, we showed in intact platelets that collagen, a PLCgamma2-dependent agonist, induces phosphorylation of PLCgamma2 at Y753 and Y759. These studies demonstrate the importance of these two tyrosine residues in regulating the activity of PLCgamma2.
