scholarly journals Ca2+-sensitive phosphoinositide hydrolysis is activated in synovial cells but not in articular chondrocytes

1999 ◽  
Vol 344 (2) ◽  
pp. 545-553 ◽  
Author(s):  
Ilaria CAPOZZI ◽  
Rossana TONON ◽  
Paola d'ANDREA
Keyword(s):  
Phospholipase C ◽  
Connexin 43 ◽  
Articular Chondrocytes ◽  
Inositol Phosphate ◽  
Receptor Activation ◽  
Phosphoinositide Hydrolysis ◽  
Functional Coupling ◽  
Synovial Cells ◽  
Dependent Manner ◽  
Concentration Dependent Manner

Cell-to-cell diffusion of second messengers across intercellular channels allows tissues to co-ordinate responses to extracellular stimuli. Intercellular diffusion of inositol 1,4,5-trisphosphate, locally produced by focal stimulations, sustains the propagation of intercellular Ca2+ waves, by stimulating the release of intracellular Ca2+ in neighbouring cells. We previously demonstrated that in cultured articular chondrocytes and HIG-82 synovial cells, studied with digitial fluorescence video imaging, mechanical stimulation of a single cell induced intercellular Ca2+ waves dependent on the presence of gap junctions. In the absence of extracellular Ca2+ the propagating distance of the wave decreased significantly in HIG-82 cells, but appeared unaffected in chondrocytes. We now show that both cells types express connexin 43 and a similar functional coupling, thus suggesting that the different Ca2+ sensitivity of intercellular waves is not due to major differences in gap junction constituent proteins. In HIG-82 synoviocytes, but not in chondrocytes, the Ca2+ ionophore ionomycin stimulated phosphoinositide hydrolysis in a concentration-dependent manner, an effect strictly dependent on the presence of extracellular Ca2+, suggesting the expression, in these cells, of a Ca2+-sensitive phospholipase C activity. Such an activity could be stimulated also by Ca2+ influx induced by P2Y receptor activation and considerably amplifies ATP-induced inositol phosphate (InsP) production. In contrast, Ca2+ influx did not affect considerably the response of chondrocytes to ATP stimulation. In HIG-82 cells, the combined application of ionomycin and ATP maximally stimulated InsP synthesis, suggesting the involvement of two independent mechanisms in inositol phosphate generation. These results suggest that in HIG-82 synovial cells the recruitment of a Ca2+-sensitive phospholipase C activity could amplify the cell response to a focally applied extracellular stimulus, thus providing a positive feedback mechanism for intercellular wave propagation.

Augmented phosphoinositide metabolism in aortas from genetically hypertensive rats

1990 ◽  
Vol 258 (1) ◽  
pp. H173-H178 ◽  
Author(s):  
M. B. Turla ◽  
R. C. Webb
Keyword(s):  
Phospholipase C ◽  
Vascular Reactivity ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Second Messengers ◽  
Receptor Activation ◽  
Phosphoinositide Hydrolysis ◽  
Phosphoinositide Metabolism ◽  
Hypertensive Rats ◽  
Wistar Kyoto

Recent studies suggest that serotonergic receptor activation is coupled to phospholipase C-mediated phosphoinositide hydrolysis, which results in the release of intracellular second messengers. The purpose of this study was to determine whether altered phosphoinositide metabolism is the basis for augmented vascular responsiveness to serotonin in genetic hypertension. Thoracic aortic segments isolated from stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto normotensive rats (WKY) were labeled with myo-[3H]inositol and stimulated with serotonin in the presence of LiCl. Accumulation of [3H]inositol phosphates was then quantitated by column chromatography. Basal inositol phosphate accumulation and basal incorporation of myo-[3H]inositol into aortic cell membranes from SHRSP was not significantly different from WKY values. At 2.6 x 10(-7) to 2.6 x 10(-4) M serotonin, phosphoinositide metabolism was significantly augmented in aortae from SHRSP compared with WKY. Depolarization (100 mM KCl) did not increase phosphoinositide hydrolysis above basal levels in SHRSP or WKY. 2-Nitro-4-carboxyphenyl-N,N-diphenyl carbamate (NCDC), an inhibitor of phospholipase C, prevented the serotonin-induced phosphoinositide metabolism. NCDC also partially inhibited phasic contractions (responses in calcium-free solution) to serotonin in aortas from SHRSP and WKY. In conclusion, abnormal phosphoinositide metabolism may be one mechanism responsible for the characteristic increase in vascular reactivity to serotonin in hypertension.

Trimucytin: A Collagen-Like Aggregating Inducer Isolated from Trimeresurus mucrosquamatus Snake Venom

1993 ◽  
Vol 69 (03) ◽  
pp. 286-292 ◽  
Author(s):  
Che-Ming Teng ◽  
Feng-Nien Ko ◽  
Inn-Ho Tsai ◽  
Man-Ling Hung ◽  
Tur-Fu Huang
Keyword(s):  
Platelet Aggregation ◽  
Snake Venom ◽  
Inositol Phosphate ◽  
Shape Change ◽  
Platelet Activating Factor ◽  
Atp Release ◽  
Platelet Membrane ◽  
Thromboxane B2 ◽  
Dependent Manner ◽  
Concentration Dependent Manner

SummaryTrimucytin is a potent platelet aggregation inducer isolated from Trimeresurus mucrosquamatus snake venom. Similar to collagen, trimucytin has a run of (Gly-Pro-X) repeats at the N-terminal amino acids sequence. It induced platelet aggregation, ATP release and thromboxane formation in rabbit platelets in a concentration-dependent manner. The aggregation was not due to released ADP since it was not suppressed by creatine phosphate/creatine phosphokinase. It was not either due to thromboxane A2 formation because indomethacin and BW755C did not have any effect on the aggregation even thromboxane B2 formation was completely abolished by indomethacin. Platelet-activating factor (PAF) was not involved in the aggregation since a PAF antagonist, kadsurenone, did not affect. However, RGD-containing peptide triflavin inhibited the aggregation, but not the release of ATP, of platelets induced by trimucytin. Indomethacin, mepacrine, prostaglandin E1 and tetracaine inhibited the thromboxane B2 formation of platelets caused by collagen and trimucytin. Forskolin and sodium nitroprusside inhibited both platelet aggregation and ATP release, but not the shape change induced by trimucytin. In quin-2 loaded platelets, the rise of intracellular calcium concentration caused by trimucytin was decreased by 12-O-tetradecanoyl phorbol-13 acetate, imipramine, TMB-8 and indomethacin. In the absence of extracellular calcium, both collagen and trimucytin caused no thromboxane B2 formation, but still induced ATP release which was completely blocked by R 59022. Inositol phosphate formation in platelets was markedly enhanced by trimucytin and collagen. MAB1988, an antibody against platelet membrane glycoprotein Ia, inhibited trimucytinand collagen-induced platelet aggregation and ATP release. However, trimucytin did not replace the binding of 125I-labeled MAB1988 to platelets. Platelets pre-exposed to trimucytin were resistant to the second challenge with trimucytin itself or collagen. It is concluded that trimucytin may activate collagen receptors on platelet membrane, and cause aggregation and release mainly through phospholipase C-phosphoinositide pathway.

scholarly journals Calcium-sensitivity of inositol 1,4,5-trisphosphate metabolism in exocrine cells from the avian salt gland

1992 ◽  
Vol 282 (3) ◽  
pp. 703-710 ◽  
Author(s):  
J P Hildebrandt ◽  
T J Shuttleworth
Keyword(s):  
Substrate Concentration ◽  
Inositol Phosphates ◽  
Receptor Activation ◽  
Dependent Manner ◽  
Salt Glands ◽  
Concentration Dependent Manner ◽  
Intact Cells ◽  
Exocrine Cells ◽  
Tissue Homogenates

The generation of inositol phosphates upon muscarinic-receptor activation was studied in [3H]inositol-loaded exocrine cells from the nasal salt glands of the duck Anas platyrhynchos, and the metabolism of different inositol phosphates in vitro was studied in tissue homogenates, with particular reference to the possible interaction of changes in intracellular [Ca2+] ([Ca2+]i) with the metabolic processes. In intact cells, there was a rapid (within 15 s) generation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4, followed by an accumulation of their breakdown products, Ins(1,3,4)P3 and inositol bis- and monophosphates. Ca(2+)-sensitivity of the Ins(1,4,5)P3 3-kinase was demonstrated in tissue homogenates, with the rate of phosphorylation increasing 2-fold at free Ca2+ concentrations greater than 1 microM. However, addition of calmodulin or the presence of the calmodulin inhibitor W-7 (up to 100 microM) had no effect. 3-Kinase activity increased proportionally with the initial Ins(1,4,5)P3 concentration up to 1 microM, but a 10-fold higher substrate concentration produced only a doubling in the phosphorylation rate. Ins(1,3,4,5)P4 was dephosphorylated to Ins(1,3,4)P3, which accumulated in the homogenate assays as well as in intact cells. Depending on its concentration, Ins(1,3,4)P3 was phosphorylated [in part to Ins(1,3,4,6)P4] or dephosphorylated. To investigate the Ca(2+)-sensitivity of the 3-kinase in intact cells, excess quin2 was used to buffer the receptor-mediated transient changes in [Ca2+]i in [3H]inositol-loaded cells. These experiments revealed that increasing [Ca2+]i from less than 100 to approx. 400 nM (i.e. within the physiological range) has no effect on the partitioning of Ins(1,4,5)P3 metabolism (phosphorylation versus dephosphorylation) and on the accumulation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4. This indicates that activation of the 3-kinase by physiologically relevant Ca2+ concentrations may not play a major role in the generation of Ins(1,3,4,5)P4 signals upon receptor activation in these cells. The latter are mainly achieved by the receptor-mediated increase in Ins(1,4,5)P3 in the cell and its phosphorylation by the 3-kinase in a substrate-concentration-dependent manner.

Effect of glucocorticoid on prostaglandin F2α-induced prostaglandin E2 synthesis in osteoblast-like cells: inhibition of phosphoinositide hydrolysis by phospholipase C as well as phospholipase A2

1994 ◽  
Vol 131 (5) ◽  
pp. 510-515 ◽  
Author(s):  
Osamu Kozawa ◽  
Haruhiko Tokuda ◽  
Atsushi Suzuki ◽  
Jun Kotoyori ◽  
Yoshiaki Ito ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase A2 ◽  
Phospholipase C ◽  
Phospholipase A ◽  
Phosphoinositide Hydrolysis ◽  
Prostaglandin E ◽  
Dependent Manner ◽  
Kinase C ◽  
Dose Dependent

Kozawa O, Tokuda H, Suzuki A, Kotoyori J, Ito Y, Oiso Y. Effect of glucocorticoid on prostaglandin F2α-induced prostaglandin E2 synthesis in osteoblast-like cells: inhibition of phosphoinositide hydrolysis by phospholipase C as well as phospholipase A2. Eur J Endocrinol 1994;131:510–15. ISSN 0804–4643 It is well known that osteoporosis is a common complication of patients with glucocorticoid excess. We showed previously that prostaglandin (PG) F2α stimulates the synthesis of PGE2, a potent bone resorbing agent, and that the activation of protein kinase C amplifies the PGF2α-induced PGE2 synthesis through the potentiation of phospholipase A2 activity in osteoblast-like MC3T3-E1 cells. In the present study, we examined the effect of dexamethasone on PGE2 synthesis induced by PGF2α in MC3T3-E1 cells. The pretreatment with dexamethasone significantly inhibited the PGE2 synthesis in a dose-dependent manner in the range between 0.1 and 10 nmol/l in these cells. This effect of dexamethasone was dependent on the time of pretreatment up to 8 h. Dexamethasone also inhibited PGE2 synthesis induced by melittin, known as a phospholipase A2 activator. Furthermore, dexamethasone significantly inhibited the enhancement of PGF2α- or melittin-induced PGE2 synthesis by 12-O-tetradecanoylphorbol-13-acetate, known as a protein kinase C activator. In addition, dexamethasone significantly inhibited PGF2α-induced formation of inositol phosphates in a dose-dependent manner between 0.1 and 10 nmol/l in MC3T3-E1 cells. These results strongly suggest that glucocorticoid inhibits PGF2α-induced PGE2 synthesis through the inhibition of phosphoinositide hydrolysis by phospholipase C as well as phospholipase A2 in osteoblast-like cells. Osamu Kozawa, Department of Biochemistry, Institute for Developmental Research, Aichi Prefectural Colony, Kasugai, Aichi 480-03, Japan

Regulation of Na(+)-3HCO3- cotransport in rabbit proximal convoluted tubule via adenosine A1 receptor

1993 ◽  
Vol 265 (4) ◽  
pp. F511-F519 ◽  
Author(s):  
M. Takeda ◽  
K. Yoshitomi ◽  
M. Imai
Keyword(s):  
Basolateral Membrane ◽  
Receptor Activation ◽  
Proximal Convoluted Tubule ◽  
Adenosine A1 Receptor ◽  
Intracellular Camp ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Adenosine A1 ◽  
A1 Receptor

We investigated the role of adenosine A1-receptor in the regulation of basolateral Na(+)-3HCO3- cotransporter in the rabbit proximal convoluted tubule (PCT) microperfused in vitro by monitoring basolateral membrane potential and intracellular pH. FK-453, a highly specific A1 antagonist, inhibited basolateral HCO3- conductance in a concentration-dependent manner (10(-10)-10(-5) M). Other A1 antagonists, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) at 10(-5) M and theophylline at 10(-3) M, also had similar effects. N6-cyclohexyladenosine (CHA) at 10(-7) M attenuated the effect of low concentration (10(-8) M) of FK-453. Either enhancement of the degradation of adenosine by 0.1 U/ml adenosine deaminase (ADA) or inhibition of adenosine release from the cells by 10(-6) M S-(4-nitrobenzyl)-6-thioinosine (NBTI) mimicked the effects of A1 antagonists. These observations suggest that endogenous adenosine is released from PCT cells and stimulates Na(+)-3HCO3- cotransporter. Both 10(-4) M 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (CPT-cAMP) and 10(-6) M forskolin also inhibited basolateral HCO3- conductance. Both 10(-6) M FK-453 and 10(-4) M CPT-cAMP decreased the initial rate as well as the magnitude of intracellular acidification induced by reduction of peritubular HCO3- concentration from 25 to 0 mM. Neither 10(-6) M FK-453 nor 10(-7) M CHA changed intracellular Ca2+ concentration as measured by fura-2 fluorescence. These results indicate that adenosine might stimulate HCO3- exit across the basolateral membrane through Na(+)-3HCO3- cotransporter by decreasing intracellular cAMP via A1-receptor activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Potentiation of angiotensin II-stimulated vascular contraction by lithium

1995 ◽  
Vol 268 (5) ◽  
pp. H2009-H2016
Author(s):  
M. E. Ullian ◽  
L. G. Walsh ◽  
K. C. Wong ◽  
C. J. Allan
Keyword(s):  
Angiotensin Ii ◽  
Inositol Phosphate ◽  
Cytosolic Calcium ◽  
Rat Aorta ◽  
Dependent Manner ◽  
Ang Ii ◽  
Concentration Dependent Manner ◽  
Adrenergic Agents ◽  
Phosphoinositide Signaling ◽  
Protein Kinase C Inhibitor

Previous studies have suggested that lithium prolongs or enhances vascular contractions stimulated by alpha-adrenergic agents. The present study was performed to determine whether a similar phenomenon occurs with angiotensin II (ANG II)-stimulated contractions and whether this phenomenon results from interactions with the phosphoinositide signaling system. Contractions of rat aortic rings with 100 nM ANG II were 38% greater in the presence of 20 mM LiCl than in its absence (0.47 +/- 0.07 vs. 0.34 +/- 0.05 g tension/mg dry tissue wt, P < 0.01). The effects of lithium on inositol phosphate responses, diacylglycerol responses, and intracellular calcium concentration on single or repeated stimulations with ANG II were then examined in vascular smooth muscle cells cultured from rat aorta. Cells exposed twice to 100 nM ANG II contained 50% lower inositol trisphosphate levels (InsP3) and 10% lower diacylglycerol levels than cells exposed to ANG II only once. LiCl or lithium acetate abolished these desensitizations in a concentration-dependent manner. Similarly, InsP3 and diacylglycerol responses to a single exposure of ANG II were heightened by lithium (by 75 and 25%, respectively), and the duration of the responses was prolonged by lithium (5- and 2-fold, respectively). In contrast, ANG II-stimulated calcium transients were not enhanced or prolonged by lithium, nor was desensitization of ANG II-stimulated cytosolic calcium mobilization upon serial exposures abolished by lithium. When ring contraction studies were repeated in the presence of the protein kinase C inhibitor staurosporine (150 nM), lithium no longer potentiated ANG II contractions [0.38 +/- 0.03 (control) vs. 0.35 +/- 0.06 g tension/mg dry tissue wt (lithium)].(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Presynaptic GABAB Receptors Regulate Retinohypothalamic Tract Synaptic Transmission by Inhibiting Voltage-Gated Ca2+ Channels

2006 ◽  
Vol 95 (6) ◽  
pp. 3727-3741 ◽  
Author(s):  
Mykhaylo G. Moldavan ◽  
Robert P. Irwin ◽  
Charles N. Allen
Keyword(s):  
Glutamate Release ◽  
Receptor Activation ◽  
Gabab Receptors ◽  
Channel Blockers ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Epsc Amplitude ◽  
Receptor Modulation ◽  
Joint Application ◽  
Retinohypothalamic Tract

Presynaptic GABAB receptor activation inhibits glutamate release from retinohypothalamic tract (RHT) terminals in the suprachiasmatic nucleus (SCN). Voltage-clamp whole cell recordings from rat SCN neurons and optical recordings of Ca2+-sensitive fluorescent probes within RHT terminals were used to examine GABAB-receptor modulation of RHT transmission. Baclofen inhibited evoked excitatory postsynaptic currents (EPSCs) in a concentration-dependent manner equally during the day and night. Blockers of N-, P/Q-, T-, and R-type voltage-dependent Ca2+ channels, but not L-type, reduced the EPSC amplitude by 66, 36, 32, and 18% of control, respectively. Joint application of multiple Ca2+ channel blockers inhibited the EPSCs less than that predicted, consistent with a model in which multiple Ca2+ channels overlap in the regulation of transmitter release. Presynaptic inhibition of EPSCs by baclofen was occluded by ω-conotoxin GVIA (≤72%), mibefradil (≤52%), and ω-agatoxin TK (≤15%), but not by SNX-482 or nimodipine. Baclofen reduced both evoked presynaptic Ca2+ influx and resting Ca2+ concentration in RHT terminals. Tertiapin did not alter the evoked EPSC and baclofen-induced inhibition, indicating that baclofen does not inhibit glutamate release by activation of Kir3 channels. Neither Ba2+ nor high extracellular K+ modified the baclofen-induced inhibition. 4-Aminopyridine (4-AP) significantly increased the EPSC amplitude and the charge transfer, and dramatically reduced the baclofen effect. These data indicate that baclofen inhibits glutamate release from RHT terminals by blocking N-, T-, and P/Q-type Ca2+ channels, and possibly by activation of 4-AP–sensitive K+ channels, but not by inhibition of R- and L-type Ca2+ channels or by Kir3 channel activation.

Effects of estrogen on cerebral blood flow and pial microvasculature in rabbits

2000 ◽  
Vol 279 (3) ◽  
pp. H1208-H1214 ◽  
Author(s):  
M. T. Littleton-Kearney ◽  
D. M. Agnew ◽  
R. J. Traystman ◽  
P. D. Hurn
Keyword(s):  
Blood Flow ◽  
Estrogen Receptor ◽  
Cerebral Blood Flow ◽  
Receptor Activation ◽  
Receptor Subtypes ◽  
Dependent Manner ◽  
Pial Arteries ◽  
Concentration Dependent Manner ◽  
Cranial Window ◽  
Pial Arterioles

We tested the hypothesis that intracarotid estrogen infusion increases cerebral blood flow (CBF) in a concentration-dependent manner and direct application of estrogen on pial arterioles yields estrogen receptor-mediated vasodilation. Rabbits of both genders were infused with estrogen via a branch of the carotid artery. Estrogen doses of 20 or 0.05 μg · ml−1 · min−1 were used to achieve supraphysiological or physiological plasma estrogen levels, respectively. CBF and cerebral vascular resistance were determined at baseline, during the infusion, and 60-min postinfusion, and effects on pial diameter were assessed via a cranial window. Pial arteriolar response to estrogen alone and to estrogen after administration of tamoxifen (10−7), an antiestrogen drug that binds to both known estrogen receptor subtypes, was tested. No gender differences were observed; therefore, data were combined for both males and females. Systemic estrogen infusion did not increase regional CBF. Estradiol dilated pial arteries only at concentrations ranging from 10−4–10−7 M ( P ≤ 0.05). Pretreatment with tamoxifen alone had no effect on arteriolar diameter but inhibited estrogen-induced vasodilation ( P < 0.001). Our data suggest that estrogen does not increase CBF under steady-state conditions in rabbits. In the pial circulation, topically applied estradiol at micromolar concentrations dilates vessels. The onset is rapid and dependent on estrogen receptor activation.

Carvedilol-induced elevation in cytosolic free Ca2+ level and apoptosis in SIRC corneal epithelial cells

2009 ◽  
Vol 29 (6) ◽  
pp. 477-487
Author(s):  
Pochuen Shieh ◽  
Chih-Hung Lee ◽  
Ng Ling Yi ◽  
Chung-Ren Jan
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Epithelial Cells ◽  
Phospholipase C ◽  
Dependent Manner ◽  
Corneal Epithelial Cells ◽  
Concentration Dependent Manner ◽  
Corneal Epithelial ◽  
Kinase C

The effect of the cardiovascular drug carvedilol on cytosolic free Ca2+ concentrations ([Ca 2+]i) and viability was examined in Statens Seruminstitut rabbit cornea (SIRC) corneal epithelial cells. [Ca2+]i and cell viability were measured using the fluorescent dyes fura-2 and 4-[3-[4-lodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate] (WST-1), respectively. Carvedilol at concentrations between 1 and 30 μM increased [Ca 2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. Carvedilol induced Mn2+ quench of fura-2 fluorescence implicating Ca2+ influx. The Ca2+ influx was inhibited by suppression of protein kinase C activity. In Ca2+-free medium, after pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca 2+ pump inhibitor), carvedilol-induced [Ca2+]i rise was reduced; and conversely, carvedilol pretreatment inhibited a major part of thapsigargin-induced [Ca 2+]i rise. Addition of the phospholipase C inhibitor 1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino] hexyl]-1H-pyrrole-2,5-dione (U73122; 2 μM) did not change carvedilol-induced [Ca2+]i rise. At concentrations between 5 and 70 μM, carvedilol killed cells in a concentration-dependent manner. The cytotoxic effect of 20 μM carvedilol was not reversed by prechelating cytosolic Ca2+ with BAPTA/AM. Apoptosis was induced by 5—70 μM carvedilol. Collectively, in SIRC corneal epithelial cells, carvedilol-induced [Ca2+]i rises by causing Ca2+ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca 2+ influx via protein kinase C-regulated Ca2+ channels. Carvedilol-caused cytotoxicity was mediated by Ca2+-independent apoptosis in a concentration-dependent manner.

Mapping the Protease Activated Receptor 4 (PAR4) Homodimer Interface.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 773-773
Author(s):  
Marvin T Nieman
Keyword(s):  
Conflicts Of Interest ◽  
Specific Interaction ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Activation ◽  
Dependent Manner ◽  
Intracellular Loop ◽  
Concentration Dependent Manner ◽  
Platelet Surface

Abstract Abstract 773 Thrombin activates platelets by binding and cleaving protease activated receptors 1 and 4 (PAR1 and PAR4). PAR1 and PAR4 communicate with each other to lower the concentration of thrombin required for PAR4 activation (Nieman Biochemistry, 2008). In addition, PAR1 and PAR4 form homo and heterodimers. However, where these receptors interact has not been defined and it is not known if dimerization influences receptor activation, downstream signaling, or both. Since PAR4 activation is important on human and mouse platelets, we sought to characterize the interaction site between PAR4 homodimers. Using bioluminescence resonance energy transfer (BRET), we mapped the PAR4 homodimer interface. The PAR4 homodimers show a specific interaction as indicated by a hyperbolic BRET signal in response to increasing PAR4-GFP expression with a fixed concentration of PAR4-Rluc. The threshold maximum BRET signal was disrupted in a concentration-dependent manner by unlabeled PAR4. In contrast, the unrelated G-protein coupled receptor, rhodopsin, was unable to disrupt the BRET signal indicating that the disruption of the PAR4 homodimer is a specific interaction. We have mapped the region required for PAR4 homodimer formation using chimeras between rhodopsin and PAR4. PAR4 does not interact with rhodopsin in BRET assays. Using a library of rho-PAR4 chimeras that have the junction at the beginning of transmembrane (TM) 2, 3, 4, 5, 6 or 7, we determined where dimer formation is restored. When the junction is placed at the beginning of TM4 or TM5, the chimera does not interact with PAR4-WT. In contrast, when the junction is moved to the end of TM2, the BRET signal is restored. These results indicate that the region on PAR4 required for homodimer formation encompasses a 63 amino acid region that includes the first extracellular loop, TM3 and the second intracellular loop. These studies establish techniques that may be used to define the interactions between other GPCRs found on the platelet surface. These receptor-receptor interactions may be another level of regulation of agonist activity and platelet function in vivo and may provide novel targets for anti-platelet therapies. Disclosures: No relevant conflicts of interest to declare.

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