Research Article: RNA sequencing to determine the contribution of kinase receptor transactivation to G protein coupled receptor signalling in vascular smooth muscle cells

Date Published: July 18, 2017

Publisher: Public Library of Science

Author(s): Danielle Kamato, Venkata Vijayanand Bhaskarala, Nitin Mantri, Tae Gyu Oh, Dora Ling, Reearna Janke, Wenhua Zheng, Peter J Little, Narin Osman, Arun Rishi.


G protein coupled receptor (GPCR) signalling covers three major mechanisms. GPCR agonist engagement allows for the G proteins to bind to the receptor leading to a classical downstream signalling cascade. The second mechanism is via the utilization of the β-arrestin signalling molecule and thirdly via transactivation dependent signalling. GPCRs can transactivate protein tyrosine kinase receptors (PTKR) to activate respective downstream signalling intermediates. In the past decade GPCR transactivation dependent signalling was expanded to show transactivation of serine/threonine kinase receptors (S/TKR). Kinase receptor transactivation enormously broadens the GPCR signalling paradigm. This work utilizes next generation RNA-sequencing to study the contribution of transactivation dependent signalling to total protease activated receptor (PAR)-1 signalling. Transactivation, assessed as gene expression, accounted for 50 percent of the total genes regulated by thrombin acting through PAR-1 in human coronary artery smooth muscle cells. GPCR transactivation of PTKRs is approximately equally important as the transactivation of the S/TKR with 209 and 177 genes regulated respectively, via either signalling pathway. This work shows that genome wide studies can provide powerful insights into GPCR mediated signalling pathways.

Partial Text

G protein coupled receptors (GPCRs) are the most prolific and polyfunctional receptors in biology [1, 2]. GPCRs control diverse physiological functions which include, relaxation of blood vessels, acceleration of heart rate, transmission of sight impulses, biorhythms and olfaction [3]. Drugs targeting GPCRs are the largest class of therapeutic agents for diseases including but not limited to: cardiovascular disease, cancer, hypertension and asthma [4–8]. The current paradigm of GPCR signalling covers three major pathways: firstly the well-defined classic signalling pathway which agonist engagement causes G protein binding to the receptor with subsequent downstream signalling leading to functional responses [9–11]. Secondly, the β-arrestin scaffold pathway which leads to the activation of multiple downstream signalling cascades [12]. Two decades ago, the GPCR signalling pathways were complemented by the finding that GPCRs could activate protein tyrosine kinase receptors (PTKRs) leading to the activation of the downstream signalling pathways [13]. This pathway termed transactivation, is the third major signalling pathway of GPCR which was recently expanded to include GPCR mediated transactivation of serine/threonine kinase receptors (S/TKR) [14–18].

RNAse-Free DNase set and miRNeasy kit were purchased from Qiagen. Dynabeads® mRNA DIRECT™ Micro kit, Ion PI™ Template OT2 Supplies 200 kit v2, Ion PI™ Template OT2 Reagents 200 Kit v2, Ion PI™ Template OT2 Solutions 200 kit v2, Ion PI™ Sequencing Supplies 200, Ion PI™ Sequencing reagents 200, Ion PI™ Sequencing solutions 200 v2, Ion PI™ Chip preparation solution and Ion Proton™ Chip Adapter where purchased from Life Technologies.

GPCRs are one of the most successful class of drug targets on the human genome for the treatment of a variety of diseases [86, 87]. Hence the role of GPCR signalling is of great interest to medical research. GPCRs signal in three main mechanisms, which includes signalling via G proteins to secondary messenger intermediates and via utilization of the signalling pathway and via the utilization of a β-arrestin molecule both which have been extensively reviewed [19, 88]. The third GPCR signalling pathway is via transactivation of PTKR and via transactivation of S/TKR. We subsequently described S/TKR transactivation but again the contribution to overall GPCR signalling was unknown. Hence in this present study, the aim was to determine the contribution of dual transactivation of GPCR mediated signalling by utilizing RNA sequencing to facilitate the identification of the genes regulated by these two transactivation pathways. Using this approach, we have uncovered that thrombin mediated signalling results in 293 differentially expressed genes of these genes approximately 50% of the signalling is occurring via transactivation dependent signalling. Using this approach we have also uncovered that transactivation of the TGFBR1 is as equally important as the transactivation of the EGFR noting that the TGFBR1 transactivation pathway has not yet been as extensively researched.




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