G protein-coupled receptors mediate the ability of extracellular hormones to regulate the activity of a wide variety of effector molecules in the cell. However, a general feature of many signalling systems is that they rapidly lose their hormonal responsiveness following receptor activation. While multiple mechanisms play a role in this loss of responsiveness, recent evidence supports a significant role of stimulus-dependent receptor phosphorylation. This phosphorylation is mediated by specific G protein-coupled receptor kinases (GRKs), such as rhodopsin kinase and the beta-adrenergic receptor kinase, the specifically phosphorylate the activated form of the receptor. While multiple GRKs have been identified, questions concerning the overall number and diversity of the GRKs, their specificity for receptor binding, and the structural features that mediate GRK/receptor interaction remain. The major objectives of this proposal are to elucidate the molecular mechanisms involved in GRK/receptor interaction and further define the role that GRKs play in mediating receptor desensitization. Multiple approaches will be used to elucidate the role of GRKs in receptor regulation. Initial studies will involve overexpression of the GRKs for purification and assessment of activity in in vitro systems. Additional studies will focus on the interactions of various receptor/GRK combinations in transiently and stably transfected cell lines. A major emphasis will be placed on elucidating the role of endogenous GRKs in intact cells. Cell lines that express the different GRKs will initially be identified using specific antibodies. Intact cell functional assays for the GRKs (e.g. agonist-induced translocation) will be developed and used to assess GRK/receptor interaction. In addition, stable transfection of dominant negative mutant GRKs and antisense cDNA constructs will be used to manipulate specific GRK levels in cells and the resulting effects on receptor desensitization will be studied. To elucidate structural features important for GRK function various mutant and chimeric GRK cDNAs will be constructed and their activity and specificity will be assessed in vitro. Additional studies will focus on the receptor domains involved in GRK binding. Potential candidate receptor domains will either be targeted by mutagenesis and/or expressed as fusion proteins and the ability of the mature receptors to interact with the various GRKs will be assessed in vitro. To further define the nature of GRK action we will also elucidate the cellular and tissue localization of the GRKs. The localization of the different GRKs in mammalian cells will be assessed by immunofluorescence using GRK-specific antibodies while in situ hybridization and immunohistochemistry will be performed to determine the tissue localization of the various GRKs. Finally, we will continue our search for additional members of the GRK gene family using the techniques of low stringency hybridization and the polymerase chain reaction. Full length clones will be isolated, sequenced, and then further characterized. These studies should further define the critical role that GRKs play in regulating hormonal responsiveness.
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