G Protein-coupled Receptor Kinase 5 In The Embryonic Hematopoietic Process The Role Of Research And Its Interacting Protein Screening

G protein-coupled receptor (GPCR) is the greatest family of membrane receptors. It plays a key role in transducing broad extracellular signals including hormones, chemokines, neurotransmitters and other environment stimuli into the intracellular. G protein-coupled receptor kinases (GRKs) are known as a large family of serine/threonine kinase that regulate GPCR signaling. GRKs interact with the agonist-activated GPCRs to catalyze receptor phosphorylation and subsequently leads to the internalization and desensitization of GPCRs.In mammalian, the GRKs consist of seven members(GRK1～7), which can be divided into three subfamilies based on the sequence and functional similarities:(1) the rhodopsin kinase subfamily (GRK1 and 7); (2) theβ-adrenergic receptor kinase subfamily (GRK2 and 3) and (3) the GRK4 subfamily (GRK4,5 and 6). GRK5, one of the most characterized members, is widely distributed in a variety of organs, especially high expression in the heart and lung, and plays important role in regulating a variety of physiological and pathological processes.Previous researches about GRKs mainly focused on the maturely developed organs. Metaye et al. observed an increase in GRK2 activity and a significant decrease in GRK5 expression in differentiated thyroid carcinoma (DTC). Knockdown GRK5 in osteosarcoma cells inhibits DNA damage-induced apoptosis via a p53-mediated mechanism. Furthermore, GRK5, but not GRK2 or GRK6, phosphorylates p53 at Thr-55, which promotes the degradation of p53, leading to inhibition of p53-dependent apoptotic response to genotoxic damage. In recent years, the role of GRKs in embryonic development has attracted much more attention. Studies with GRK2 knockout mice have shown that GRK2 plays an important role in embryonic cardiac development and function, No GRK2-/- embryos survive beyond gestational day 15.5 because of cardiac malformations. Our latest research demonstrates that GRK2 interacts with PTCH1, increases the nuclear translocation of cyclin B1 and promotes cyclin B1 mediated cell cycle and embryonic development. Based on the above researches, the role of GRK5 in mouse embryonic development was investigated in this thesis. Our results showed that:1. GRK5 knockout mice displayed impaired blood-island. Hematoxylin-Eosin staining were performed on E8.5 embryo sections of WT and GRK5 knockout mice and revealed that the normal blood-island can be detected in WT and GRK5 knockout mice embryos were 71% and 27% respectively; the embryos size of GRK5 knockout mice was smaller than WT mice. These results imply that GRK5 may play role in the embryonic hematopoiesis development.2. Results of co-immunopriciptation and GST-pull down indicated that GRK5 can interact with GATA2 in vivo and in vitro.3. Since the blood-island impaired in the embryo of GRK5 knockout mice, we tested the peripheral blood of adult mice and found that the numbers of red blood cells and platelets were not affected while the white blood cells were significantly decreased in GRK5 knockout mice, and this decrease mainly because of the decrease of granulocytes.4. Immunoprecipitation-Mass spectrography was performed to investigate the proteins interact with GRK5. These proteins can be grouped into 3 categories:RNA splicing protein; DNA damage repair complex such and cytoskeleton related protein. We also validated the results of IP-MS through immunoprecipitation.Above all, our research indicate that (1) GRK5 is important for the embryonic hematopoiesis development. (2) GRK5 can interact with GATA2 in vitro. (3) GRK5 knockout mice showed reduced white blood cells in peripheral blood. (4) GRK5 may play important roles in other unknown processes though interacting with other proteins. Our research may provide a new idea for further research of the mechanism of GRK5 in mouse embryonic development and other processes.