Effect Of G Protein - Coupled Receptor Kinase 5 (GRK5) On Angiogenesis And Its Mechanism

G protein-coupled receptors(GPCRs), the largest family of transmembrane receptors, play a key role in transducing broad extracellular signals from diverse stimulations, including neuromediators, glycoproteinic hormones, sensory substances, as well as a large variety of small molecules, thus regulatemultiple biological processes.G protein-coupled receptor kinases (GRKs) are key regulators of GPCR function.Phosphorylation-specific GRKs play a key role in triggering rapid receptor internalization and desensitization. There are7members in the GRK family, which can be categorized into3subfamilies. GRK5is ubiquitously distributed in multiple organs, and is found most abundantly expressed in heart, lung,brain, muscle and peripheral tissues. To this day, a variety of GPCRs have been foundregulated by GRK5selectively, and GRK5plays important roles in mediating physiological functions as well as pathological processes. Besides its well-known role as a negative regulator of GPCRs, some recent evidence has also shown additional physiological significance of GRK5.Our current study demonstrated the effect of GRK5on angiogenesis. Angiogenesis, the growth of new blood vessels,is an essential process duringdevelopment, adulthoodwound healing and female menstrual cycle. Angiogenesis is regulated by a very sensitive interplayof growth factors and inhibitors, the imbalance of which can result in serious diseases.Our current study revealed a novel function of GRK5in the regulation of angiogenesis, and investigated the effect of GRK5on the mechanism of action inendothelial cells during angiogenesis.Our results showed that:1. GRK5is highly expressed in endothelial cells in different human and mouse organs. GRK5knockout (GRK5-/-) mice showed severe defectsin angiogenic sprouting and vascular remodelingat mid-gestation. Knockdown of GRK5in zebrafish embryos caused irregular development of zebrafish trunk vascular. The developmental deficiencies could be rescued by wild type GRK5or kinase dead mutant K215R, indicating the regulatory functions of GRK5on embryonic developmental angiogenesis.2. We found reduced tumor growth in GRK5-/-mice independent of cancer type or tumourlocation. A consistent histologicalfinding in tumors of GRK5-/-mice were a markedly lower densityof microvessels and an elevated tumor necrosis. Time on wound healing of GRK5-/-mice is extended. All the results indicate that GRK5deletion impairs pathologicalangiogenesis.3. Angiogenesis is a process begining with endothelial cell activation by VEGF and other pro-angiogenic factors. We found the VEGF-contained matrigel plug implanted in GRK5-/-mice was less likely to trigger mice endothelial cells than wildtype mice. The primary endothelial cells from GRK5-/-were also deficient in monolayer migration, VEGF-induced migration, capillary-like tube formation in vitro. All these results indicate that GRK5can regulate angiogenic response and endothelial cell migration.4. We found that GRK5depletion caused membrane ruffle reduction and stress fiber expansion in endothelia cells, which indicated GTPase RhoA up-regulation. RhoA inhibitor C3and ROCK inhibitor Y27632blocked cell phenotype alterations caused by GRK5RNAi. And GRK5depletion caused increased focal adhension of endothelial cell. The results indicate that GRK5can down regulate RhoA-ROCK signaling and regulates endothelial cells migration.5. We employed biochemistry assay and detected co-ordinated changes of RhoA activation after GRK5protein level alteration. And we also found GRK5binding of GDP-RhoA. These results indicate that the regulation of RhoA activation by GRK5may act through GRK5locking of inactivated RhoA from its effector.6. VEGF stimulation or mechanical stretch can induce membrane ruffle formation in endothelial cells. We found GRK5localized in these membrane ruffle. By biochemistry assay we found GRK5translocated to membrane after VEGF stimulation. And its membrane-unbound and F-actin-unbound mutants did not cause stress fiber reduction and membrane ruffle formation. The mutants also could not rescue developmental angiogenic defects by grk5knockdown in zebrafish embryos. These results indicate that GRK5regulates RhoA activation in endothelial cells by pro-angiogenic factor, and promotes angiogenesis.Our studies were the first to find the regulatory function of GRK5in angiogenesis. GRK5translocates from cytosol to membrane by the stimulation of VEGF; blocks the activation type of GTPase RhoA probably through their interaction. These implicate the mechanism of GRK5on regulating endothelial cells migration, promoting tumor angiogenesis and developmental angiogenesis. Our data reveal novel function of GRK5on angiongenesis, which may supply as potential drug target for clinical use in vascular related disease.