Role In Regulation Of G Protein-coupled Receptor Kinase In Embryonic Development

G protein-coupled receptors (GPCRs), the greatest 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 regulate a wide variety of biological processes. GPCRs have already become potential drug targets in various diseases. G protein-coupled receptor kinases (GRKs) are key regulatory kinases for GPCRs. Receptor phosphorylation by specific GRKs plays a key role in triggering rapid internalization and desensitization. There are 7 members in the GRK family (GRK1-7), which can be categorized into 3 subfamilies. Being the best-characterized members, GRK2 and GRK5 ubiquitously distribute in multiple organs, and play important roles in mediating a variety of physiological functions as well as pathological processes. GRK2 and GRK5 share structural conservations in their catalytic domain. However, they show structural as well as functional differences in many aspects. For example, GRK2 is the only member of GRK family, whose gene ablation results in lethal effects in mice, and the mechanism still remains unclear. Because of the nuclear locating sequence (NLS), GRK5 and its subfamily colleagues distribute in the whole cell, including the cytoplasm and the cell nuclei, while other GRKs only show up in the cytoplasm; however, the nuclear functions of GRK5 is still poorly understood.Previous research mainly focuses on the regulatory functions of GRK2 and GRK5 in differentiated cells and maturely developed organs. Roles of GRKs in the dynamic process of embryonic development were poorly defined. On the basis of understanding roles of key regulators, such as GRKs, in embryonic development, different drug effects on adults and fetals would be better elucidated, and it would be helpful to prevent developmental abnormalities.In our research, we applied the animal model of zebrafish for the developmental study of GRKs, and depending on the developmental phenotypes of zebrafish embryos and mechanism explorations on human cells, we revealed de novo roles of GRKs in regulating embryonic development. Our results showed that:1. Both GRK2 and GRK5 expressed at high levels in zebrafish early-staged embryos. Knockdown of either GRK2 or GRK5 caused embryonic developmental deficiencies. GRK2 knockdown resulted in developmental early arrest and cell cycle arrest in G2/M stage, while knockdown of GRK5 mainly influenced the process of embryonic hematopoiesis. The developmental deficiencies could be rescued not only by wild type GRK2 or GRK5, but also by their kinase dead mutants K220R or K215R, indicating that the regulatory functions of GRKs on embryonic early development might not rely on their kinase activity.2. Results of yeast-two hybrid screening suggested that PTCH1, the regulator of cyclin B1, might be a binding partner of GRK2. Results of the microarray suggested that the hematopoietic transcriptional factor GATA2 might be regulated by GRK5. Here we tested in different cells the interaction of GRK2 and PTCH1, as well as the interaction of GRK5 and GATA2, and found out their binding sites, respectively. We found that BP, the binding domain of GRK2 with PTCH1, showed rescue effect in the developmental arrest and cell cycle arrest in GRK2 knockdown embryos and cells, but the binding deficiency mutant A312-379 did not. R216L, the binding deficiency mutant of GRK5 with GATA2, could not rescue the hematopoietic abnormality in GRK5 knockdown embryos; what's more, M130, the peptide containing the binding site, even showed dominant negative effects in embryonic hematopoiesis. These results demonstrated that the interaction of GRK2 and PTCH1, and the interaction of GRK5 and GATA2 are essential in embryonic developmental regulation.3. Hedgehog (ShhN), the agonist of PTCH1, promoted the association of GRK2 and PTCH1, and reduced the association of PTCH1 and cyclin B1. GRK2, K220R, as well as BP, promoted the dissociation of cyclin B1 from PTCH1, increased the translocation of cyclin B1 into the cell nuclei, but A312-379 did not. These results suggested that the interaction between GRK2 and PTCH1, which was stimulated by the Hedgehog agonist, might be the potential mechanism for the regulation of GRK2 on the cyclin pathway. Binding of GRK2 with PTCH1 reduced the interaction of cyclin B1 and PTCH1, disrupted PTCH1-mediated inhibition of cyclin B1 nuclear translocation, and promoted cyclin B1-mediated cell cycle, cell proliferation and embryonic development.4. GRK5, K215R, as well as the nuclear-located GRK2 mutant NLS-GRK2 promoted the nuclear accumulation of GATA2, and enhanced its transcriptional activity, but R216L or the nuclear locating deficiency mutant of GRK5, NES-GRK5, did not, indicating that the nuclear localization of GRK5 and its interaction with GATA2 are both essential in accumulating GATA2 in the cell nuclei and promoting the transctiptional activity of GATA2.Above all, our research disclosed important roles of GRK2 and GRK5 in embryonic development, and found out their regulatory mechanisms, respectively. GRK2 interacted with PTCH1, the regulator of cyclin B1, induced the dissociation of cyclin B1 and PTCH1, increased the nuclear translocation of cyclin B1, and promoted cyclin B1 mediated cell cycle and embryonic development. GRK5 interacted with GATA2, accumulated GATA2 in the cell nuclei, enhanced the transcriptional activity of GATA2, and thus promoted the differentiation of hematopoietic stem cells and the embryonic hematopoietic process. Our data thus revealed novel regulatory functions of GRKs in embryonic development and the kinase activity-independent mechanisms underlying their regulatory functions.