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Research has been conducted on the G protein-coupled receptors. The study of the nonreceptor substrates of these receptors has been carried out via the two-dimensional electrophoresis and the use of this electrophoresis in tracking cellular proteins phosphorylated after agonist-stimulation of the beta (sub)2 -adrenergic receptor in a G protein-coupled receptor kinase 2 manner is described.

Results point out that elongation factor G and eukaryotic elongation factor 2 evolved separately and the latter acquired an ATP/ADP binding site along with its phosphorylation and ADP-ribosylation sites. Data further reveal two different sites binding either guanylic or adenylic nucleotides.

Characterizes the G Protein-Coupled Receptor (GPCR) kinases for the ability to phosphorylate and desensitize GPCR. Phosphorylation of tubulin by receptor kinases; Identification of ribosomal protein P2 as HEK-293 cell protein; Role of GPCR and tranlational control of gene expression on phosphorylation in animals.

G protein-coupled receptor kinases are well characterized for their ability to phosphorylate and desensitize G protein-coupled receptors (GPCRs). In addition to phosphorylating the beta2-adrenergic receptor (beta2AR) and other receptors, G protein-coupled receptor kinase 2 (GRK2) can also phosphorylate tubulin, a nonreceptor substrate. To identify novel nonreceptor substrates of GRK2, we used two-dimensional gel electrophoresis to find cellular proteins that were phosphorylated upon agonist-stimulation of the beta2AR in a GRK2-dependent manner. The ribosomal protein P2 was identified as an endogenous HEK-293 cell protein whose phosphorylation was increased following agonist stimulation of the beta2AR under conditions where tyrosine kinases, PKC and PKA, were inhibited. P2 along with its other family members, P0 and P1, constitutes a part of the elongation factor-binding site connected to the GTPase center in the 60S ribosomal subunit. Phosphorylation of P2 is known to regulate protein synthesis in vitro. Further, P2 and P1 are shown to be good in vitro substrates for GRK2 with K(M) values approximating 1 microM. The phosphorylation sites in GRK2-phosphorylated P2 are identified (S102 and S105) and are identical to the sites known to regulate P2 activity. When the 60S subunit deprived of endogenous P1 and P2 is reconstituted with GRK2-phosphorylated P2 and unphosphorylated P1, translational activity is greatly enhanced. These findings suggest a previously unrecognized relationship between GPCR activation and the translational control of gene expression mediated by GRK2 activation and P2 phosphorylation and represent a potential novel signaling pathway responsible for P2 phosphorylation in mammals.