Joseph N. McLaughlin

Protease-activated receptor-3 (PAR3) regulates PAR1 signaling by receptor dimerization

Thrombin activates endothelial cell signaling by cleaving the protease-activated receptor-1 (PAR1). However, the function of the apparently nonsignaling receptor PAR3 also expressed in endothelial cells is unknown. We demonstrate here the crucial role of PAR3 in potentiating the responsiveness of PAR1 to thrombin. We tested the hypothesis that PAR1/PAR3 heterodimerization and its effect in modifying G protein selectivity was responsible for PAR3 regulation of PAR1 sensitivity. Using bioluminescent resonance energy transfer-2, we showed that PAR1 had comparable dimerization affinity for PAR3 as for itself. We observed increased Gα13 coupling between the PAR1/3 heterodimer compared with the PAR1/1 homodimer. Moreover, knockdown of PAR3 moderated the PAR1-activated increase in endothelial permeability. These results demonstrate a role of PAR3 in allosterically regulating PAR1 signaling governing increased endothelial permeability. Because PAR3 is a critical determinant of PAR1 function, targeting of PAR3 may mitigate the effects of PAR1 in activating endothelial responses such as vascular inflammation.

PAR4, but not PAR1, signals human platelet aggregation via Ca2+ mobilization and synergistic P2Y12 receptor activation

Regulation of platelet activation plays a central role in hemostasis and pathophysiological processes such as coronary artery disease. Thrombin is the most potent activator of platelets. Human platelets express two thrombin receptors, PAR1 and PAR4, both of which signal platelet activation. Evidence is lacking on the mechanism by which PAR1 and PAR4 may differentially signal platelet aggregation. Here we show that at the relatively high concentration of agonist most likely found at the site of a local thrombus, dual inhibition of the P2Y12 receptor and calcium mobilization result in a complete inhibition of PAR4-induced aggregation, while having no effect on either thrombin or PAR1-mediated platelet aggregation. Both PAR1- and PAR4mediated aggregation are independent of calcium mobilization. Furthermore, we show that P2Y12 receptor activation is not required for protease-activated receptor-mediated aggregation at higher agonist concentrations and is only partially required for Rap1 as well as GPIIbIIIa activation. P2Y12 receptor inhibitors clinically in use such as clopidogrel are postulated to decrease platelet aggregation through partial inhibition of PAR1 signaling. Our data, however, indicate that at high local concentrations of thrombin, it is the signaling through PAR4 rather than PAR1 that may be regulated through purinergic feedback. Thus, our data identify an intra-platelet mechanism that may function as a future site for therapeutic intervention.

Phosducin-like protein acts as a molecular chaperone for G protein βγ dimer assembly

Phosducin‐like protein (PhLP) is a widely expressed binding partner of the G protein βγ subunit dimer (Gβγ). However, its physiological role is poorly understood. To investigate PhLP function, its cellular expression was blocked using RNA interference, resulting in inhibition of Gβγ expression and G protein signaling. This inhibition was caused by an inability of nascent Gβγ to form dimers. Phosphorylation of PhLP at serines 18–20 by protein kinase CK2 was required for Gβγ formation, while a high‐affinity interaction of PhLP with the cytosolic chaperonin complex appeared unnecessary. PhLP bound nascent Gβ in the absence of Gγ, and S18–20 phosphorylation was required for Gγ to associate with the PhLP‐Gβ complex. Once Gγ bound, PhLP was released. These results suggest a mechanism for Gβγ assembly in which PhLP stabilizes the nascent Gβ polypeptide until Gγ can associate, resulting in membrane binding of Gβγ and release of PhLP to catalyze another round of assembly.

A molecular mechanism for the phosphorylation-dependent regulation of heterotrimeric G proteins by phosducin.

Visual signal transduction is a nearly noise-free process that is exquisitely well regulated over a wide dynamic range of light intensity. A key component in dark/light adaptation is phosducin, a phosphorylatable protein that modulates the amount of transducin heterotrimer (Gt alpha beta gamma) available through sequestration of the beta gamma subunits (Gt beta gamma). The structure of the phosphophosducin/Gt beta gamma complex combined with mutational and biophysical analysis provides a stereochemical mechanism for the regulation of the phosducin-Gt beta gamma interaction. Phosphorylation of serine 73 causes an order-to-disorder transition of a 20-residue stretch, including the phosphorylation site, by disrupting a helix-capping motif. This transition disrupts phosducins interface with Gt beta gamma, leading to the release of unencumbered Gt beta gamma, which reassociates with the membrane and Gt alpha to form a signaling-competent Gt alpha beta gamma heterotrimer.

Regulatory interaction of phosducin-like protein with the cytosolic chaperonin complex

Phosducin and phosducin-like protein (PhLP) bind G protein βγ subunits and regulate their activity. This report describes a previously uncharacterized binding partner unique to PhLP that was discovered by coimmunoprecipitation coupled with mass spectrometric identification. Chaperonin containing tailless complex polypeptide 1 (CCT), a cytosolic chaperone responsible for the folding of many cellular proteins, binds PhLP with a stoichiometry of one PhLP per CCT complex. Unlike protein-folding substrates of CCT, which interact only in their nonnative conformations, PhLP binds in its native state. Native PhLP competes directly for binding of protein substrates of CCT and thereby inhibits CCT activity. Overexpression of PhLP inhibited the ability of CCT to fold newly synthesized β-actin by 80%. These results suggest that the interaction between PhLP and CCT may be a means to regulate CCT-dependent protein folding or alternatively, to control the availability of PhLP to modulate G protein signaling.

PAR1, but Not PAR4, Activates Human Platelets through a Gi/o/Phosphoinositide-3 Kinase Signaling Axis

Thrombin-mediated activation of platelets is critical for hemostasis, but the signaling pathways responsible for this process are not completely understood. In addition, signaling within this cascade can also lead to thrombosis. In this study, we have defined a new signaling pathway for the thrombin receptor protease activated receptor-1 (PAR1) in human platelets. We show that PAR1 couples to Gi/o in human platelets and activates phosphoinositide-3 kinase (PI3K). PI3K activation regulates platelet integrin αIIbβ3 activation and platelet aggregation and potentiates the PAR1-mediated increase in intraplatelet calcium concentration. PI3K inhibitors eliminated these effects downstream of PAR1, but they had no effect on PAR4 signaling. This study has identified an important role for the direct activation of Gi/o by PAR1 in human platelets. Given the efficacy of clopidogrel, which blocks the Gi/o-coupled P2Y purinoceptor 12, as an antiplatelet/antithrombotic drug, our data suggest that specifically blocking only PAR1-mediated Gi/o signaling could also be an effective therapeutic approach with the possibility of less unwanted bleeding.

Modulation of the G Protein Regulator Phosducin by Ca2+/Calmodulin-dependent Protein Kinase II Phosphorylation and 14-3-3 Protein Binding

Phototransduction is a canonical G protein-mediated cascade of retinal photoreceptor cells that transforms photons into neural responses. Phosducin (Pd) is a Gβγ-binding protein that is highly expressed in photoreceptors. Pd is phosphorylated in dark-adapted retina and is dephosphorylated in response to light. Dephosphorylated Pd binds Gβγ with high affinity and inhibits the interaction of Gβγ with Gα or other effectors, whereas phosphorylated Pd does not. These results have led to the hypothesis that Pd down-regulates the light response. Consequently, it is important to understand the mechanisms of regulation of Pd phosphorylation. We have previously shown that phosphorylation of Pd by cAMP-dependent protein kinase moderately inhibits its association with Gβγ. In this study, we report that Pd was rapidly phosphorylated by Ca2+/calmodulin-dependent kinase II, resulting in 100-fold greater inhibition of Gβγ binding than cAMP-dependent protein kinase phosphorylation. Furthermore, Pd phosphorylation by Ca2+/calmodulin-dependent kinase II at Ser-54 and Ser-73 led to binding of the phosphoserine-binding protein 14-3-3. Importantly, in vivodecreases in Ca2+ concentration blocked the interaction of Pd with 14-3-3, indicating that Ca2+ controls the phosphorylation state of Ser-54 and Ser-73 in vivo. These results are consistent with a role for Pd in Ca2+-dependent light adaptation processes in photoreceptor cells and also suggest other possible physiological functions.

Functional roles of the two domains of phosducin and phosducin-like protein.

Phosducin and phosducin-like protein regulate G protein signaling pathways by binding the βγ subunit complex (Gβγ) and blocking Gβγ association with Gα subunits, effector enzymes, or membranes. Both proteins are composed of two structurally independent domains, each constituting approximately half of the molecule. We investigated the functional roles of the two domains of phosducin and phosducin-like protein in binding retinal Gtβγ. Kinetic measurements using surface plasmon resonance showed that: 1) phosducin bound Gtβγ with a 2.5-fold greater affinity than phosducin-like protein; 2) phosphorylation of phosducin decreased its affinity by 3-fold, principally as a result of a decrease in k 1; and 3) most of the free energy of binding comes from the N-terminal domain with a lesser contribution from the C-terminal domain. In assays measuring the association of Gtβγ with Gtα and light-activated rhodopsin, both N-terminal domains inhibited binding while neither of the C-terminal domains had any effect. In assays measuring membrane binding of Gtβγ, both the N- and C-terminal domains inhibited membrane association, but much less effectively than the full-length proteins. This inhibition could only be described by models that included a change in Gtβγ to a conformation that did not bind the membrane. These models yielded a free energy change of +1.5 ± 0.25 kcal/mol for the transition from the Gtα-binding to the Pd-binding conformation of Gtβγ.

Regulation of Angiotensin II-induced G Protein Signaling by Phosducin-like Protein

Phosducin-like protein (PhLP) is a broadly expressed member of the phosducin (Pd) family of G protein βγ subunit (Gβγ)-binding proteins. Though PhLP has been shown to bind Gβγ in vitro, little is known about its physiological function. In the present study, the effect of PhLP on angiotensin II (Ang II) signaling was measured in Chinese hamster ovary cells expressing the type 1 Ang II receptor and various amounts of PhLP. Up to 3.6-fold overexpression of PhLP had no effect on Ang II-stimulated inositol trisphosphate (IP3) formation, whereas further increases caused an abrupt decrease in IP3 production with half-maximal inhibition occurring at 6-fold PhLP overexpression. This threshold level for inhibition corresponds to the cellular concentration of cytosolic chaperonin complex, a recently described binding partner that preferentially binds PhLP over Gβγ. Results of pertussis toxin sensitivity, GTPγS binding, and immunoprecipitation experiments suggest that PhLP inhibits phospholipase Cβ activation by dual mechanisms: (i) steric blockage of Gβγ activation of PLCβ and (ii) interference with Gβγ-dependent cycling of Gqα by the receptor. These results suggest that G protein signaling may be regulated through controlling the cellular concentration of free PhLP by inducing its expression or by regulating its binding to the chaperonin.