Mapping Human Protease-activated Receptor 4 (PAR4) Homodimer Interface to Transmembrane Helix 4

Fuente, María de la; Noble, Daniel N.; Verma, Sheetal; Nieman, Marvin T.

doi:10.1074/jbc.m112.341438

Cited by 36 publications

(61 citation statements)

References 63 publications

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“…Molecular Cloning-The human PAR4 and the PAR4 transmembrane helix 4 mutants have been described (see Table 1) (15). PAR4 with a mutation at the P1 residue of the thrombin cleavage site (PAR4-R47Q) was generated with overlapping PCR as previously described (18).…”

Section: Methodsmentioning

confidence: 99%

“…Bioluminescence Resonance Energy Transfer (BRET)-Initial experiments determined the optimal expression of PAR1 for BRET experiments as previously described for PAR4 (15). HEK293 cells (1 ϫ 10 5 ) were transfected with PAR1-GFP (0 -1 g) or PAR1-rLuc (0 -0.5 g) to determine the minimal amount of plasmid for sufficient GFP and luciferase signal.…”

Section: Methodsmentioning

confidence: 99%

“…The conditions that gave optimal expression for BRET studies were used in subsequent experiments. BRET experiments were performed as previously described (15). To account for lower expression of PAR1, all PAR1-rLuc constructs were adjusted to 0.5 g/transfection.…”

Section: Methodsmentioning

confidence: 99%

“…To account for lower expression of PAR1, all PAR1-rLuc constructs were adjusted to 0.5 g/transfection. PAR4-rLuc was used at 0.03 g/transfection as previously described (15). The PAR4-GFP constructs were used at 0 -0.25 g/transfection, and the PAR1-GFP constructs were used at 0 -5 g/transfection.…”

Section: Methodsmentioning

confidence: 99%

“…For example, PAR4 activation produces a prolonged signal that is required for stable clot formation (11)(12)(13)(14). Our laboratory has recently mapped the PAR4 homodimer interface to transmembrane helix 4 (15). Mutations that disrupt PAR4 homodimers also disrupt Ca 2ϩ signaling.…”

mentioning

confidence: 99%

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Protease-activated Receptor 1 (PAR1) and PAR4 Heterodimers Are Required for PAR1-enhanced Cleavage of PAR4 by α-Thrombin

Arachiche

Mumaw

Fuente

et al. 2013

Journal of Biological Chemistry

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Background: Protease-activated receptor 1 (PAR1) and PAR4 mediate thrombin signaling in platelets. Results: Mutations in transmembrane helix 4 (TM4) of PAR1 or PAR4 disrupts ␣-thrombin-induced heterodimerization and PAR1-assisted PAR4 cleavage. Conclusion: PAR1-PAR4 heterodimers are required for efficient PAR4 cleavage. Significance: The dimerization of PAR1 and PAR4 may impact the effectiveness of PAR1 antagonists.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Protease-activated Receptor 1 (PAR1) and PAR4 Heterodimers Are Required for PAR1-enhanced Cleavage of PAR4 by α-Thrombin

Arachiche

Mumaw

Fuente

et al. 2013

Journal of Biological Chemistry

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Protease receptor antagonism to target blood platelet therapies

Holinstat

Bray

2015

Clin Pharma and Therapeutics

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Platelet activation and thrombus formation play a central role in ischemic vascular disease. Thrombin, an especially potent physiologic agonist mediating in vivo activation of platelets, acts via a unique family of G-protein-coupled receptors called protease-activated receptors (PARs) with a broad tissue expression. This review focuses on current antiplatelet therapies as well as innovative approaches to targeting PARs in patients with atherothrombotic vascular disease.

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Murine cadherin‐6 mediates thrombosis in vivo in a platelet‐independent manner

Bouck

Fuente

Zunica

et al. 2021

Research and Practice in Thrombosis and Haemostasis

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Background Platelet adhesion is the critical process mediating stable thrombus formation. Previous reports of cadherin‐6 on human platelets have demonstrated its role in platelet aggregation and thrombus formation. Objectives We aimed to further characterize the importance of cadherin‐6 in thrombosis in vivo. Methods Cadherin‐6 platelet expression was evaluated by western blotting, flow cytometry, and immunoprecipitation. Thrombosis was evaluated using the FeCl 3 and Rose Bengal carotid artery models in C57Bl6 mice treated with anti–cadherin‐6 or IgG and wild‐type or Cdh6 −/− mice. Platelet function was compared in wild‐type and Cdh6 −/− mice using tail‐clip assays, aggregometry, and flow cytometry. Results Human platelet expression of cadherin‐6 was confirmed at ~3000 copies per platelet. Cdh6 −/− mice or those treated with anti–cadherin‐6 antibody showed an increased time to occlusion in both thrombosis models. Cadherin‐6 was not expressed on mouse platelets, and there were no differences in tail bleeding times, platelet aggregation, or platelet activation in wild‐type versus Cdh6 −/− mice. Conclusions Cadherin‐6 plays an essential role in thrombosis in vivo. However, cadherin‐6 is not expressed on murine platelets. These data are in contrast to human platelets, which express a functional cadherin‐6/catenin complex. The essential, platelet‐independent role for cadherin‐6 in hemostasis may allow it to be an effective and safe therapeutic target.

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Mapping Human Protease-activated Receptor 4 (PAR4) Homodimer Interface to Transmembrane Helix 4

Cited by 36 publications

References 63 publications

Protease-activated Receptor 1 (PAR1) and PAR4 Heterodimers Are Required for PAR1-enhanced Cleavage of PAR4 by α-Thrombin

Protease-activated Receptor 1 (PAR1) and PAR4 Heterodimers Are Required for PAR1-enhanced Cleavage of PAR4 by α-Thrombin

Protease receptor antagonism to target blood platelet therapies

Murine cadherin‐6 mediates thrombosis in vivo in a platelet‐independent manner

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