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

Abstract

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.

A cellular perspective of bias at G protein‐coupled receptors

“…Microarrays and RNAseq provide an alternative approach to more holistically capture how GPCR activation changes the cell. Such approaches show that GPCR signaling can activate the transcription of hundreds of genes 39,62–64 . As a point of comparison, activation of RTKs can induce changes to the transcription of ~1,000 genes 65–67 .…”

GPCR transactivation signalling in vascular smooth muscle cells: role of NADPH oxidases and reactive oxygen species

“…We previously found that the GPCR agonist thrombin transactivates the EGFR and TGFBR1 to stimulate the expression of enzymes involved in the hyperelongation of glycosaminoglycan chains on the proteoglycan, biglycan ( 123 , 124 ) which is associated with increased lipid retention in the vessel wall initiating atherosclerosis ( 125 , 126 ). We have described that GPCR transactivation of either receptor is occurring via completely different mechanisms and the identification of a common mechanism can attenuate all GPCR-mediated GAG chain elongation ( 127 , 128 ). However, established data for GPCR transactivation of PTKRs and newly emerging data for mechanisms of S/TKRs indicates that ROS may be involved in both transactivation mechanisms and as such ROS would represent the first common mechanism and hence the first potential target to prevent all transactivation signalling.…”

QuAdTrim: Overcoming computational bottlenecks in sequence quality control

“…QuAdTrim was first developed in 2011 for use in a metagenomic study that utilised Illumina paired end sequence data (Ross, et al, 2012). Subsequently QuAdTrim has been used for quality control of Illumina sequence data in from human, bacterial, virus, cattle, sheep, and salmon studies amoung others (Bolormaa, et al, 2019;Daetwyler, et al, 2017;Kamato, et al, 2017;Kijas, et al, 2019;Kijas, et al, 2018;Wei, et al, 2018).…”