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Title: Association of type 1 inositol 1,4,5-trisphosphate receptor (IP₃R1) with protein kinase A and A-kinase Anchoring Protein 9 (AKAP9) in platelets
Author: Trivedi, Arti N.
ISNI:       0000 0004 6346 7783
Awarding Body: University of Hull and University of York
Current Institution: University of Hull
Date of Award: 2017
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Cyclic adenosine monophsphoate (cAMP) signalling is thought to regulate inositol-1,4,5 trisphosphate (IP₃)-mediated calcium (Ca²⁺) mobilisation in platelets through protein kinase A (PKA)-mediated phosphorylation, leading to inhibition of inositol-1,4,5 trisphosphate receptors (IP₃Rs). A- kinase Anchoring Proteins (AKAPs) are known to be involved in spatial and temporal regulation of PKA-mediated phosphorylation; however the precise molecular mechanisms regulating the PKA-mediated inhibition of IP₃Rs remain to be fully understood. To further understand this, the presence and functional importance of a novel PKA/IP₃R/AKAP signalling complex in platelets was studied. Protein complexes identified using co-immunoprecipitation and analysed using immunoblotting, while Ca²⁺ measurements were made using spectrofluorometry. We show that platelet stimulation with thrombin evoked a sharp increase in intracellular Ca²⁺ levels, which was blocked by IP₃R1 antagonist, 2-Aminoethoxydiphenyl borate (2-APB). This suggested Ca²⁺ mobilisation through IP₃Rs. Furthermore, activation of cAMP signalling by prostacyclin (PGI2)-treatment restricted Ca²⁺ mobilisation in response to thrombin treatment, suggesting that an involvement of cAMP pathway in regulation of IP₃R channel activity. Furthermore, the presence of all three isoforms of IP₃R in platelets was confirmed by immunoblotting. PGI2-induced phosphorylation of IP₃R1 on Serine1756 (Ser1756) was blocked by pharmacological inhibitors of PKA, and mimicked by direct activators of PKA and adenylyl cyclase. Immunoprecipitates of IP₃R1 also showed an associated PKA activity, owing to the coimmunoprecipitation of PKA with IP₃R1. The functional importance of this association was explored using a cell-permeable peptide PKA-AKAP disruptor peptide; St-Ht31. St-Ht31 inhibited PGI2-induced phosphorylation of IP₃R1 and blunted the ability of PGI2 to inhibit the Ca²⁺ mobilisation. This suggests a role of AKAPs in regulation of Ca²⁺ mobilisation. Numerous AKAPs have been putatively identified in platelets through transcriptomics and proteomics studies. Here, we show the presence of AKAP9 in platelets. Moreover, using coimmunoprecipitation, the association of AKAP9 with PKA/IP₃R1 was also suggested. These data suggest the presence of a novel PKA/IP₃R1/AKAP9 signalling complex in platelets that may modulate intracellular Ca²⁺ mobilisation.
Supervisor: Naseem, Khalid ; Calaminus, Simon Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Medicine