G Protein-coupled Receptor Kinase 5 Regulatory Role In The Development Of Zebrafish Hematopoietic System

G protein-coupled receptor (GPCR) family represents the largest class of cell surface receptors with seven transmembrane domains.It can transduce a large number of extracellular signals including hormones, neurotransmitters, chemokins and other environmental stimuli to the intracellular compartment. Widely involved in various physiological functions, itself is regulated exquisitly through a variety of ways, such as GPCR desensitization through G protein-coupled receptor kinase(GRK)catalyzing the phosphorylation of agonist-occupied GPCR. Seven different members of GRKs have been found for now:GRK1-7.Their distribution and substrate are not very similar.Hematopoietic system development is controled by a series of transcription factors, GATA family transcription factors play key roles in this process.GATA2 is expressed at the stem and progenitor stage, including erythroid lineage, in hematopoiesis, is essential for hematopoietic system development. Studies of our lab and the new researchs have shown that GRKs play an important role in zebrafish embryo development through interaction with PTCH1 and phorphorylating Smo.In this study, on the basis of previous work, taking zebrafish as a research model, we initially explored the role of G protein-coupled receptor kinase 5 on the regulation of hematopoietic system development and its mechanism.Our results show that:(1)Based on the GRK5 homologous sequences in zebrafish gene pool, we extracted zebrafish total RNA and cloned the full-length sequence of zebrafish GRK5 utilizing RACE-PCR method. (2) We taked a preliminary study of GRK5's distribution in zebrafish through Whole-mount In Situ Hybridization. Knockdown GRK5 in zebrafish through morpholino reduced the expression of Fli-1,Gata1--two transcription factors related to hematopoietic system development, this down-expression can be rescued by GRK5 and GRK5-K215R. This results indicate that GRK5 may play roles in hematopoiesis.(3)We initialy found the interaction between GRK5 and GATA2 by coimmunoprecipitation approach, and the 216 arginine of GRK5 is essential to this interaction. (4) We overexpressed GRK5 or knockdown GRK5 in K562 cells and then screened the GATA2 downstream genes related to hematopoietic system utilizing Real Time PCR, found that the expression of a-globin,γ-globin, MLF1, P27 and PU.1 is changed obviously. Further experiments found that GRK5 mutant R216L can not rescue the down-expression of mark gene related to hematopoiesis by knockdown of GRK5 in zebrafish.In conclusion, our researchs indicate that(1)GRK5 is required for the normal expression of marker genes related to zebrafish hematopoietic development, indicating that GRK5 is required for hematopoiesis.(2) GRK5 interacts with GATA2 and regulates the expression of GATA2 downstream genes through interaction with GATA2, this results provide a new idea for further research of the regulation mechanism of GRK5 on the development of zebrafish hematopoiesis.