G protein coupled receptor transactivation of kinase receptors: the new signalling frontier

Kamato, D 2016, G protein coupled receptor transactivation of kinase receptors: the new signalling frontier, Doctor of Philosophy (PhD), Health Sciences, RMIT University.


Document type: Thesis
Collection: Theses

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Title G protein coupled receptor transactivation of kinase receptors: the new signalling frontier
Author(s) Kamato, D
Year 2016
Abstract G-protein coupled receptors (GPCRs) are the largest class of cell surface receptors. GPCR signalling is mediated through the classical signalling pathways, and non-classical signalling via utilisation of β-arrestin molecule and the transactivation of protein tyrosine kinase receptors (PTKR) and activation of serine/threonine kinase receptors (S/TKR), most notably the transforming growth factor (TGF)-β receptor (TGFBR1). Protease-activated receptors (PARs) are a family of GPCRs that are activated by the action of thrombin. Thrombin via its GPCR, PAR-1, can transactivate both PTKR in particular the epidermal growth factor receptor (EGFR) and the S/TKR TGFBR1. Thrombin signalling in human vascular smooth muscle cells (VSMCs) has been shown to contribute to atherosclerosis. Atherosclerosis commences by the trapping of lipoproteins in the vessel wall by modified proteoglycans specifically elongated glycosaminoglycan (GAG) chains. Thrombin through the transactivation of the EGFR and TGFBR1 leads to an increase of the GAG chain lengths allowing for an increase in low density lipoprotein retention. The two transactivation dependent pathways have distinct mechanisms. The PTKR mediated response involves matrix metalloproteinases and the phosphorylation of Erk. The S/TKR mediated response differs markedly and involves the phosphorylation of Smad2 carboxy terminus. The aim of this project was to expand on the mechanisms involved in transactivation dependent signalling and to find a common signalling intermediate which can inhibit both transactivation dependent signalling pathways leading to proteoglycan synthesis.

The in vitro model used human VSMCs. GAG synthesizing gene expression was measured and quantified by real time-PCR. Signalling intermediate phospho-proteins were detected and quantified by western blotting. Proteoglycan synthesis was assessed by labelling of GAG chains with [35S]‑Sulphate and assessing incorporation into GAG chains via CPC precipitation assay. Proteoglycan GAG chain elongation was assessed by SDS‑PAGE. Next generation RNA sequencing was conducted using the Ion proton sequencer. Reads were aligned to the human genome using TopHat software and differentially expressed genes were evaluated using the edgeR software. One-way ANOVA, followed by least significance difference post hoc analysis was used for statistical significance.

Thrombin treatment of VSMCs increased the mRNA expression of GAG synthesizing genes, CHST11, CHSY1 and CHST3. Thrombin via the transactivation of the PTKR, EGFR and S/TKR, TGFBR1 regulated the mRNA expression of CHST11, CHSY1, CHST3 and biglycan. The role of Gαq was investigated in this transactivation signalling pathway by utilizing Gαq antagonist UBO-QIC. Gαq played a role in thrombin transactivation of the EGFR and TGFBR1 and was involved in thrombin mediated proteoglycan synthesis, GAG elongation and regulation of the mRNA expression of CHST11 and CHSY1. Mechanistic studies revealed that thrombin transactivation of the TGFBR1 is mediated via Gαq which leads to Rho/ROCK signalling which activates cell surface integrin αVβ5 leading to the activation of the TGFBR1. siRNA knockdown of the Gαq and Gαq/11 revealed that thrombin mediated mRNA expression of CHST11 and CHSY1 occurs specifically via Gαq but not the Gαq/11 protein.

Thrombin treatment of VSMCs mediates phosphorylation of four serine/threonine residues of the Smad2 linker region. Thrombin mediated phosphorylation of the Smad2 linker region is via the transactivation of the TGFBR1 and the EGFR. Phosphorylation of the linker region via transactivation of the EGFR occurs within 15 mins and is sustained for 4 hours. The phosphorylation of the Smad2 linker region via transactivation of the TGFBR1 is temporal initiating at 120 mins post thrombin treatment and sustained up to 4 hours. The individual serine/threonine Smad2 linker region residues were phosphorylated by either Erk, p38, PI3K or CDK, however Jnk was only involved in thrombin transactivation of the EGFR leading to the phosphorylation of the Thr220 residue. Erk, p38 PI3K and CDK are involved in regulating thrombin mediated mRNA expression of CHST11 and CHSY1.

RNA-Seq sequencing analysis revealed that in human VSMCs thrombin was involved in upregulating 293 genes. Thrombin treatment in the presence of EGFR antagonist and TGFBR1 antagonist inhibited the expression to 129 and 151 differentially expressed genes, respectively. Thrombin transactivation of the EGFR and TGFBR1 shared 140 genes. Thrombin transactivation of the EGFR regulated 512 biological processes and transactivation of the TGFBR1 regulated 428 biological processes. From the top 50 biological processes enriched by transactivation dependent signalling, 28 terms were found to be common between the two pathways. Many of the biological processes were associated with regulation of cell activity as well as vasculature development.

The findings demonstrate that thrombin via the transactivation of the EGFR and TGFBR1 regulates the mRNA expression of CHST11, CHSY1, CHST13 and biglycan. The transactivation pathways have distinct mechanisms and the Gαq protein plays as the central integrating point. The phosphorylation of the serine residues of the Smad2 linker region is most likely associated with GAG synthesizing genes involved in GAG chain elongation whereas the phosphorylation of the threonine residue of Smad2 linker region is not. Transactivation dependent signalling contributes to 50% of PAR-1 mediated signalling, with the two transactivation pathways sharing in over 60% of common differentially expressed genes. Thus showing that a central integrating point in the transactivation dependent signalling pathways may not only be essential for proteoglycan synthesis and the initiation of atherosclerosis but may help regulate the biological processes associated with the 60% common differentially expressed genes.
Degree Doctor of Philosophy (PhD)
Institution RMIT University
School, Department or Centre Health Sciences
Subjects Signal Transduction
Pharmacology and Pharmaceutical Sciences not elsewhere classified
Keyword(s) G protein coupled receptors
Transforming growth factor beta
Smad
Atherosclerosis
Transactivation
Kinase receptors
Protein tyrosine kinase receptor
serine/threonine kinase receptor
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