Signal transduction through G␣ q involves stimulation of phospholipase C␤ (PLC␤) that results in increased intracellular Ca 2؉and activation of protein kinase C. We have measured complex formation between G␣ q and PLC␤ 1 in vitro and in living PC12 and HEK293 cells by fluorescence resonance energy transfer. In vitro measurements show that PLC␤ 1 will bind to G␣ q (guanosine 5-3-O-(thio)triphosphate) and also to G␣ q (GDP), and the latter association has a different protein-protein orientation. In cells, image analysis of fluorescent-tagged proteins shows that G␣ q is localized almost entirely to the plasma membrane, whereas PLC␤ 1 has a significant cytosolic population. By using fluorescence resonance energy transfer, we found that these proteins are pre-associated in the unstimulated state in PC12 and HEK293 cells. By determining the cellular levels of the two proteins in transfected versus nontransfected cells, we found that under our conditions overexpression should not significantly promote complex formation. G␣ q -PLC␤ 1 complexes are observed in both single cell measurements and measurements of a large (i.e. 10 6 ) cell suspension. The high level (ϳ40% maximum) of FRET is surprising considering that G␣ q is more highly expressed than PLC␤ 1 and that not all PLC␤ 1 is plasma membrane-localized. Our measurements suggest a model in which G proteins and effectors can exist in stable complexes prior to activation and that activation is achieved through changes in intermolecular interactions rather than diffusion and association. These pre-formed complexes in turn give rise to rapid, localized signals.

Protein palmitoylation is a dynamic process that regulates membrane targeting of proteins and protein-protein interactions. We have previously demonstrated a critical role for protein palmitoylation in platelet activation and have identified palmitoylation machinery in platelets. Using a novel proteomic approach, Palmitoyl Protein Identification and Site Characterization, we have begun to characterize the human platelet palmitoylome. Palmitoylated proteins were enriched from membranes isolated from resting platelets using acyl-biotinyl exchange chemistry, followed by identification using liquid chromatography-tandem mass spectrometry. This global analysis identified > 1300 proteins, of which 215 met criteria for significance and represent the platelet palmitoylome. This collection includes 51 known palmitoylated proteins, 61 putative palmitoylated proteins identified in other palmitoylationspecific proteomic studies, and 103 new putative palmitoylated proteins. Of these candidates, we chose to validate the palmitoylation of triggering receptors expressed on myeloid cell (TREM)-like transcript-1 (TLT-1) as its expression is restricted to platelets and megakaryocytes. We determined that TLT-1 is a palmitoylated protein using metabolic labeling with

Pleckstrin homology (PH) domains are membrane tethering devices found in many signal transducing proteins. These domains also couple to the ␤␥ subunits of GTP binding proteins (G proteins), but whether this association transmits allosteric information to the catalytic core is unclear. To address this question, we constructed protein chimeras in which the PH domain of phospholipase C-␤ 2 (PLC-␤ 2 ), which is regulated by G␤␥, replaces the PH domain of PLC-␦ 1 which binds to, but is not regulated by, G␤␥. We found that attachment of the PH domain of PLC-␤ 2 onto PLC-␦ 1 not only causes the membrane-binding properties of PLC-␦ 1 to become similar to those of PLC-␤ 2 , but also results in a G␤␥-regulated enzyme. Thus, PH domains are more than simple tethering devices and mediate regulatory signals to the host protein.Pleckstrin homology (PH) 1 domains are ϳ100 amino acid structural modules which, despite low amino acid sequence homology (i.e. only one conserved residue), appear to have the same overall spatial topology of a barrel-like structure formed from seven ␤-sheets with one side closed off by a C-terminal ␣-helix (for review, see Ref. 1). PH domains have been identified in over 100 diverse proteins involved in cell signaling and/or membrane/cytoskeletal interactions. While it is generally agreed that PH domains help tether their host proteins to membrane surfaces, some PH domains, such as that of the ␤-adrenergic receptor kinase (2, 3), have also been implicated as binding sites for G␤␥ subunits.All mammalian inositol-specific phospholipase C (PLC) enzymes contain a PH domain at their N terminus. PLCs are soluble enzymes that catalyze the hydrolysis of a minor component in membranes, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ), releasing the two second messengers inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol (for review, see Refs. 4 and 5). These enzymes have a multidomain structure (Fig. 1) containing, besides the N-terminal PH domain, four elongation factor (EF) hands, a catalytic X-Y domain with an insertion of varying length, and a C2 domain (6). There are three known families of mammalian PLCs (␤, ␥, and ␦), each with a distinct set of protein regulators. PLC-␤ isoenzymes are activated by ␣ q and ␤␥ subunits of heterotrimeric GTP binding proteins (G proteins). PLC-␥ enzymes are regulated by receptor tyrosine kinases. Protein regulators of PLC-␦ enzymes are unknown although RhoGAP and transglutaminase have been implicated (7,8).PLCs must associate with membranes during catalysis, and their PH domain plays a key role in membrane attachment. The PH domain of PLC-␥ binds specifically to membranes containing PI(3,4,5)P 3 (9) and the PH domain of PLC-␦ binds strongly to membranes containing PI(4,5)P 2 (10, 11). In contrast, the PH domain of PLC-␤ binds strongly and nonspecifically to membranes which helps to promote its lateral association with G protein subunits, and it has been found that the isolated PH domain of PLC-␤ 2 binds to G-␤␥ on membranes with a similar strong affinity as that o...

G protein-coupled receptors (GPCRs) can assume multiple conformations and possess multiple binding sites. Whereas endogenous agonists acting at the orthosteric binding site stabilize the active receptor conformation, small molecules that act at nonorthosteric sites can stabilize alternative conformations. The large majority of these allosteric modulators associate with extracellular loops of GPCRs. The role of intracellular domains in mediating allosteric modulation is largely unknown. In screening a small-molecule library for inhibitors of platelet activation, we identified a family of compounds that modified PAR1-mediated granule secretion. The most potent inhibitory compound, termed JF5, also demonstrated noncompetitive inhibition of the α 2A -adrenergic receptor. Aggregation studies using a battery of platelet GPCR agonists demonstrated that sensitivity to JF5 was limited to GPCRs that possessed a constrained eighth helix, as defined by a C-terminal palmitoylation site and interactions with TM7 and the i1 loop. Inhibition by JF5 was overcome in a PAR1 mutant in which the eighth helix was deleted, confirming a role for helix 8 in JF5 activity. Evaluation of downstream signaling showed that JF5 was selective with regard to G protein coupling, blocking signaling mediated by G αq but not G α12 . The compound inhibited thrombus formation in vivo following vascular injury with an IC 50 of ∼1 mg/kg. These results indicate a role for helix 8 in conferring sensitivity to small molecules, and show that this sensitivity can be exploited to control platelet activation during thrombus formation.thrombosis | chemical genetics | thrombin receptor | platelet signaling