The First Intracellular Loop Regulates The Platelet Thromboxane A2 Receptor‐Gq‐Dependent Signaling And Function

Abstract

While the molecular signaling pathways for the thromboxane A2 receptor (TPR) have been elucidated from multiple organ systems, much less research efforts have focused on the TPR‐ G‐protein (GP) coupling domains (particularly in platelets). In this connection, studies conducted thus far (mostly mutagenesis in nature) have implicated regions from all of the intracellular domains of TPR in coupling to GPs. However, since alterations in the amino acid sequence can lead to effects on protein structure, at least some of these receptor alterations may not be at the critical GP coupling sites. Based on these considerations, we attempted to map the GP coupling domains and investigate human platelet signaling through TPR by employing site‐specific peptides targeted against regions potentially‐involved in the receptor‐Gα subunits interaction process. Our findings revealed that a myristoylated peptide that mimics the first intracellular loop (IL1) of human TPR (i.e., amino acids A52 ‐ F63; abbreviated as Myr‐IL1pep) blocked TPR‐mediated human and mouse platelet aggregation, but (interestingly) it had no detectable effects on platelet shape change (SC). This finding suggests that the IL1 domain of TPR participates in receptor coupling to Gαq but not Gα13. Furthermore, these data are consistent with previous findings that while platelet aggregation is a Gαq‐dependent event, SC is Gα13 dependent. The specificity of this inhibition was shown by the findings that: 1. the control random sequence for IL1 (Myr‐IL1random) did not produce any detectable effect on platelet aggregation induced by TPR activation; and 2. the Myr‐IL1pep itself did not interfere with ADP‐triggered aggregation. Regarding a potential mechanism, immunoaffinity co‐purification experiments revealed that Myr‐IL1pep uncoupled Gαq but not Gα13 from TPR, whereas the Myr‐IL1random did not produce any apparent effects. Finally, the Myr‐IL1pep was found to protect against thrombogenesis, but it does so while also prolonging the tail bleeding time, in mice; whereas control peptides were without any effect on thrombosis or bleeding time. Collectively, our results provide novel information concerning TPR's structural biology and signaling, and may identify new and novel therapeutic agents for the prevention and/or treatment of thrombotic disorders.Support or Funding InformationThis work was supported by the National Heart, Lung, And Blood Institute of the National Institutes of Health under Award Number R15HL115567 (to F.T.K). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.