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Mapping human protease-activated receptor 4 (PAR4) homodimer interface to transmembrane helix 4.

ABSTRACT: Thrombin activates platelets by binding and cleaving protease-activated receptors 1 and 4 (PAR1 and PAR4). Because of the importance of PAR4 activation on platelets in humans and mice and emerging roles for PAR4 in other tissues, experiments were done to characterize the interaction between PAR4 homodimers. Bimolecular fluorescence complementation and bioluminescence resonance energy transfer (BRET) were used to examine the PAR4 homodimer interface. In bimolecular fluorescence complementation experiments, PAR4 formed homodimers that were disrupted by unlabeled PAR4 in a concentration-dependent manner, but not by rhodopsin. In BRET experiments, the PAR4 homodimers showed a specific interaction as indicated by a hyperbolic BRET signal in response to increasing PAR4-GFP expression. PAR4 did not interact with rhodopsin in BRET assays. The threshold maximum BRET signal was disrupted in a concentration-dependent manner by unlabeled PAR4. In contrast, rhodopsin was unable to disrupt the BRET signal, indicating that the disruption of the PAR4 homodimer is not due to nonspecific interactions. A panel of rho-PAR4 chimeras and PAR4 point mutants has mapped the dimer interface to hydrophobic residues in transmembrane helix 4. Finally, mutations that disrupted dimer formation had reduced calcium mobilization in response to the PAR4 agonist peptide. These results link the loss of dimer formation to a loss of PAR4 signaling.

SUBMITTER: de la Fuente M

PROVIDER: S-EPMC3322995 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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Mapping human protease-activated receptor 4 (PAR4) homodimer interface to transmembrane helix 4.

de la Fuente María M Noble Daniel N DN Verma Sheetal S Nieman Marvin T MT

The Journal of biological chemistry 20120208 13

Thrombin activates platelets by binding and cleaving protease-activated receptors 1 and 4 (PAR1 and PAR4). Because of the importance of PAR4 activation on platelets in humans and mice and emerging roles for PAR4 in other tissues, experiments were done to characterize the interaction between PAR4 homodimers. Bimolecular fluorescence complementation and bioluminescence resonance energy transfer (BRET) were used to examine the PAR4 homodimer interface. In bimolecular fluorescence complementation ex ...[more]

PMID: 22318735

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Project description:Recently, we reported that the chemokine (C-X-C motif) receptor 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3) heteromerize with α1A/B/D-adrenoceptors (ARs) and arginine vasopressin receptor 1A (AVPR1A) in recombinant systems and in rodent and human vascular smooth muscle cells (hVSMCs). In these studies, we observed that heteromerization between two receptor partners may depend on the presence and the expression levels of other partnering receptors. To test this hypothesis and to gain initial insight into the formation of these receptor heteromers in native cells, we utilized proximity ligation assays in hVSMCs to visualize receptor-receptor proximity and systematically studied how manipulation of the expression levels of individual protomers affect heteromerization patterns among other interacting receptor partners. We confirmed subtype-specific heteromerization between endogenously expressed α1A/B/D-ARs and detected that AVPR1A also heteromerizes with α1A/B/D-ARs. siRNA knockdown of CXCR4 and of ACKR3 resulted in a significant re-arrangement of the heteromerization patterns among α1-AR subtypes. Similarly, siRNA knockdown of AVPR1A significantly increased heteromerization signals for seven of the ten receptor pairs between CXCR4, ACKR3, and α1A/B/D-ARs. Our findings suggest plasticity of seven transmembrane helix (7TM) receptor heteromerization in native cells and could be explained by a supramolecular organization of these receptors within dynamic clusters in the plasma membrane. Because we previously observed that recombinant CXCR4, ACKR3, α1a-AR and AVPR1A form hetero-oligomeric complexes composed of 2-4 different protomers, which show signaling properties distinct from individual protomers, re-arrangements of receptor heteromerization patterns in native cells may contribute to the phenomenon of context-dependent GPCR signaling. Furthermore, these findings advise caution in the interpretation of functional consequences after 7TM receptor knockdown in experimental models. Alterations of the heteromerization patterns among other receptor partners may alter physiological and pathological responses, in particular in more complex systems, such as studies on the function of isolated organs or in in vivo experiments.

Dataset Information

Mapping human protease-activated receptor 4 (PAR4) homodimer interface to transmembrane helix 4.

Publications

Mapping human protease-activated receptor 4 (PAR4) homodimer interface to transmembrane helix 4.

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