Final Activity Report Summary - DETERMINANTS OF GRK2 (Structural and functional determinants of GRK2 in cardiac development)
G protein-coupled receptor kinases (GRK) provide the rate-limiting step for terminating incoming signals at G protein-coupled receptors (GPCR). By phosphorylating the active receptor, they protect the cell from overstimulation. Extensive studies underlined the important role of GRK2, which is the most common GRK in regulating cardiac function in the adult. However, the role of GRK2, which is indispensable during murine development, was only preliminary analysed and was therefore the subject of the funded project.
The novelty of the project was based on a combined in vivo approach by using zebrafish as a second model organism instead of extrapolating in vivo relevance from in vitro or cell culture studies. To accomplish this, I received training in manipulating and analysing zebrafish and in techniques commonly used in signal transduction, such as site-directed cloning and biochemical assays, which complemented my previous research experience in mice and chicken embryos. In addition, I widened my international experience in research as well as my personal horizon.
During the funding period, I identified the signalling cascade, which was defective in mice lacking GRK2. The knockdown of the close homolog of mammalian GRK2 in zebrafish resulted in misshaped somites, brain and pericardiac edema, in an abnormally developed heart and a disrupted neural tube patterning. Using genetic and biochemical approaches in vivo and in vitro I found that GRK2 was required to phosphorylate smoothened and that, in the absence of GRK2, smoothened-mediated hedgehog (Hh) signalling was impaired in zebrafish. By site-directed mutagenesis on GRK2 I found that its function in Hh signalling depended on the kinase activity of GRK2 and its ability to bind ß? subunits of heterotrimeric G proteins. In situ hybridisation revealed expression of zebrafish GRK2 in areas adjacent to sonic hedgehog expression, which was a secreted morphogen, where smoothened, the protein transducing the Hh signal into the cell, was expressed. I found similar deficiencies in Hh signalling in mouse GRK2 KO embryos, along with a dilated heart and pericardiac edema just as in zebrafish. I concluded that GRK2 facilitated Hh signalling by phosphorylating the GPCR-like protein smoothened.
These results were specific for GRK2, as loss of both variants of GRK5 in zebrafish did not cause the same anatomical malformations nor change Hh signalling. Instead, I found a role for one of the two GRK5 variants, GRK5a, in Wnt signalling, which formed another fundamental signal pathway during embryonic development. In collaboration with professors Chen, Premont and Lefkowitz, all located in Duke University, Durham, NC, United States, we identified GRK5 as a positive modulator of Wnt signalling by phosphorylating the Wnt co-receptor LRP6. This result was even more striking, as it had always been assumed that GRKs could only phosphorylate GPCRs, which were characterised by a seven transmembrane structure, while LRP consisted by only one transmembrane domain. Using in vitro experiments accompanied by studies in cell culture and knockdown experiments in zebrafish, we could provide evidence for the involvement of GRK5 in canonical Wnt signalling.
In summary, I finished two studies describing novel, unanticipated roles of GRKs during embryonic development throughout the funding period. I also established collaborations with leading scientists in the field of receptor-mediated signal transduction. In an ongoing project, together with my supervisor at Duke University, Prof. Dr Marc G. Caron and the laboratory of Dr Arthur Moseley at Duke University, I tried to uncover for the first time the phospho-profile of different GRKs in vivo. To do so, we developed a label-free, cost-efficient platform to assess the phosphorylation state of a complex sample using mass spectrometry.
The novelty of the project was based on a combined in vivo approach by using zebrafish as a second model organism instead of extrapolating in vivo relevance from in vitro or cell culture studies. To accomplish this, I received training in manipulating and analysing zebrafish and in techniques commonly used in signal transduction, such as site-directed cloning and biochemical assays, which complemented my previous research experience in mice and chicken embryos. In addition, I widened my international experience in research as well as my personal horizon.
During the funding period, I identified the signalling cascade, which was defective in mice lacking GRK2. The knockdown of the close homolog of mammalian GRK2 in zebrafish resulted in misshaped somites, brain and pericardiac edema, in an abnormally developed heart and a disrupted neural tube patterning. Using genetic and biochemical approaches in vivo and in vitro I found that GRK2 was required to phosphorylate smoothened and that, in the absence of GRK2, smoothened-mediated hedgehog (Hh) signalling was impaired in zebrafish. By site-directed mutagenesis on GRK2 I found that its function in Hh signalling depended on the kinase activity of GRK2 and its ability to bind ß? subunits of heterotrimeric G proteins. In situ hybridisation revealed expression of zebrafish GRK2 in areas adjacent to sonic hedgehog expression, which was a secreted morphogen, where smoothened, the protein transducing the Hh signal into the cell, was expressed. I found similar deficiencies in Hh signalling in mouse GRK2 KO embryos, along with a dilated heart and pericardiac edema just as in zebrafish. I concluded that GRK2 facilitated Hh signalling by phosphorylating the GPCR-like protein smoothened.
These results were specific for GRK2, as loss of both variants of GRK5 in zebrafish did not cause the same anatomical malformations nor change Hh signalling. Instead, I found a role for one of the two GRK5 variants, GRK5a, in Wnt signalling, which formed another fundamental signal pathway during embryonic development. In collaboration with professors Chen, Premont and Lefkowitz, all located in Duke University, Durham, NC, United States, we identified GRK5 as a positive modulator of Wnt signalling by phosphorylating the Wnt co-receptor LRP6. This result was even more striking, as it had always been assumed that GRKs could only phosphorylate GPCRs, which were characterised by a seven transmembrane structure, while LRP consisted by only one transmembrane domain. Using in vitro experiments accompanied by studies in cell culture and knockdown experiments in zebrafish, we could provide evidence for the involvement of GRK5 in canonical Wnt signalling.
In summary, I finished two studies describing novel, unanticipated roles of GRKs during embryonic development throughout the funding period. I also established collaborations with leading scientists in the field of receptor-mediated signal transduction. In an ongoing project, together with my supervisor at Duke University, Prof. Dr Marc G. Caron and the laboratory of Dr Arthur Moseley at Duke University, I tried to uncover for the first time the phospho-profile of different GRKs in vivo. To do so, we developed a label-free, cost-efficient platform to assess the phosphorylation state of a complex sample using mass spectrometry.