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

Arachiche, Amal; Mumaw, Michele M.; Fuente, María de la; Nieman, Marvin T.

doi:10.1074/jbc.m113.472373

Cited by 76 publications

(78 citation statements)

References 49 publications

(78 reference statements)

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“…22,23 In each case, the enhanced rate of PAR4 cleavage by thrombin is dependent on PAR4 heterodimerization. 23,78,79 In platelets, PAR4 also forms agonist dependent heterodimers with the ADP receptor, P2Y12. 76,77 Li et al showed that simultaneous stimulation of PAR4 and P2Y12 resulted in arrestin-2 recruitment to PAR4.…”

mentioning

confidence: 99%

“…PARs are no exception as evidence exists for both constitutive and agonist-induced dimers. 8,23,[75][76][77][78] The major point of contention regarding the organization of GPCRs is defining a reliable readout for oligomerization of receptors in native tissues. PARs have a distinct advantage over other receptor families because they cooperate in specific ways that are consistent with dimerization or oligomerization.…”

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confidence: 99%

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Protease-activated receptors in hemostasis

Nieman

2016

Blood

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126

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Protease signaling in cells elicits multiple physiologically important responses via protease-activated receptors (PARs). There are 4 members of this family of G-protein–coupled receptors (PAR1-4). PARs are activated by proteolysis of the N terminus to reveal a tethered ligand. The rate-limiting step of PAR signaling is determined by the efficiency of proteolysis of the N terminus, which is regulated by allosteric binding sites, cofactors, membrane localization, and receptor dimerization. This ultimately controls the initiation of PAR signaling. In addition, these factors also control the cellular response by directing signaling toward G-protein or β-arrestin pathways. PAR1 signaling on endothelial cells is controlled by the activating protease and heterodimerization with PAR2 or PAR3. As a consequence, the genetic and epigenetic control of PARs and their cofactors in physiologic and pathophysiologic conditions have the potential to influence cellular behavior. Recent studies have uncovered polymorphisms that result in PAR4 sequence variants with altered reactivity that interact to influence platelet response. This further demonstrates how interactions within the plasma membrane can control the physiological output. Understanding the structural rearrangement following PAR activation and how PARs are allosterically controlled within the plasma membrane will determine how best to target this family of receptors therapeutically. The purpose of this article is to review how signaling from PARs is influenced by alternative cleavage sites and the physical interactions within the membrane. Going forward, it will be important to relate the altered signaling to the molecular arrangement of PARs in the cell membrane and to determine how these may be influenced genetically.

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confidence: 99%

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confidence: 99%

Protease-activated receptors in hemostasis

Nieman

2016

Blood

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126

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“…75 Additionally, affinity tag removal from the N-terminal domain of Platelet Activating Factor proteins have facilitated heterodimer formation. 76 Without the his-tag, the Δ85 IncA dimer is 42.6 kDa, and the elution pattern was consistent with expectations ( Figure 4.4). 74 SEC data presented here suggest specificity in structural alignment exists to promote dimer formation and the monomer population is produced by interference by the flexible his-tag.…”

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confidence: 74%

Cloning, Expression, and Biophysical Investigations of Truncated Inclusion Protein A (IncA) from Chlamydia trachomatis

Campbell

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ii To my mom and dad, who gave me a thirst for knowledge and the desire to learn.iii Acknowledgements I would like to thank Dr. Linda Columbus for allowing me to work in her lab, study inclusion proteins, and pushing my limits.Thank you Columbus and Mura Lab members for all their technical assistance and feedback.Dr. John D. Shannon for his incredible patience and assistance with MALDI-TOF MS and circular dichroism.Many thanks to Jessica Sarver in the Cafiso lab for allowing me to think out loud about EPR.Thank you to Sandy, Vicki, and Kevin in Facilities Management who kept me laughing, updated me on the news and "goings on" in the department in the "wee" hours of the morning.Special thanks to Tsega Solomon who gave me invaluable technical advice, interesting philosophical debates over lunch, and a renewed interest in shopping. How I will miss you! Special thanks to Dr. Kim Bassett for her listening ear and unconditional friendship. demonstrated the hexa-histidine tag disrupted dimer formation; however, after cleavage of the tag with thrombin, dimer was reformed. Circular dichroism of the construct confirmed the dimer was all α-helical as expected for a SNARE motif. Finally, two single cysteine mutants were prepared and spin-labeled. Continuous wave electron paramagnetic resonance confirmed the sites were spin-labeled and the lineshapes were consistent with moderately immobilized spin labels expected for a tertiary contact. Future double electronelectron resonance experiments will measure the distance between the spin labels, which will elucidate the dimer orientation.

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“…25,28 Although there may have been a small reduction in total receptor expression in this overexpression model, we showed that the Y157C substitution resulted in much more marked reduction of surface expression of PAR4, thereby reducing the availability of receptor to respond extracellular agonists. Consistent with this, PAR4 Y157C showed greater colocalization than PAR4 WT with the ER exit-marker calnexin, but less colocalization with early endosome antigen 1, which is a marker of endosomes that mediate GPCR internalization after surface membrane expression.…”

Section: Discussionmentioning

confidence: 93%

“…2 The preservation of PAR1-AP responses in the PAR4 Y157C platelets is less consistent with previous observations in transfected cell models that PAR1-PAR4 heterodimer formation is necessary for full PAR1 expression and function. 28 It is not possible to resolve from these functional data whether the heterozygous Y157C substitution in the cases disrupts PAR4 homodimer formation, 29 which could contribute to the loss of PAR4 responsiveness phenotype.…”

Section: Discussionmentioning

confidence: 99%

Protease-Activated Receptor 4 Variant p.Tyr157Cys Reduces Platelet Functional Responses and Alters Receptor Trafficking

Norman

Cunningham

Jones

et al. 2016

ATVB

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Objective-Protease-activated receptor 4 (PAR4) is a key regulator of platelet reactivity and is encoded by F2RL3, which has abundant rare missense variants. We aimed to provide proof of principle that rare F2LR3 variants potentially affect platelet reactivity and responsiveness to PAR1 antagonist drugs and to explore underlying molecular mechanisms. Approach and Results-We identified 6 rare F2RL3 missense variants in 236 cardiac patients, of which the variant causing a tyrosine 157 to cysteine substitution (Y157C) was predicted computationally to have the greatest effect on PAR4 structure. Y157C platelets from 3 cases showed reduced responses to PAR4-activating peptide and to α-thrombin compared with controls, but no reduction in responses to PAR1-activating peptide. Pretreatment with the PAR1 antagonist vorapaxar caused lower residual α-thrombin responses in Y157C platelets than in controls, indicating greater platelet inhibition. HEK293 cells transfected with a PAR4 Y157C expression construct had reduced PAR4 functional responses, unchanged total PAR4 expression but reduced surface expression. PAR4 Y157C was partially retained in the endoplasmic reticulum and displayed an expression pattern consistent with defective N-glycosylation. Mutagenesis of Y322, which is the putative hydrogen bond partner of Y157, also reduced PAR4 surface expression in HEK293 cells. Conclusions-Reduced

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

Cited by 76 publications

References 49 publications

Protease-activated receptors in hemostasis

Protease-activated receptors in hemostasis

Cloning, Expression, and Biophysical Investigations of Truncated Inclusion Protein A (IncA) from Chlamydia trachomatis

Protease-Activated Receptor 4 Variant p.Tyr157Cys Reduces Platelet Functional Responses and Alters Receptor Trafficking

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