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The G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate and desensitize agonist-occupied GPCRs. GRK2-mediated receptor phosphorylation is preceded by the agonist-dependent membrane association of this enzyme. Previous in vitro studies with purified proteins have suggested that this translocation may be mediated by the recruitment of GRK2 to the plasma membrane by its interaction with the free βγ subunits of heterotrimeric G proteins (Gβγ). Here we demonstrate that this mechanism operates in intact cells and that specificity is imparted by the selective interaction of discrete pools of Gβγ with receptors and GRKs. Treatment of Cos-7 cells transiently overexpressing GRK2 with a β-receptor agonist promotes a 3-fold increase in plasma membrane-associated GRK2. This translocation of GRK2 is inhibited by the carboxyl terminus of GRK2, a known Gβγ sequestrant. Furthermore, in cells overexpressing both GRK2 and Gβ 1 γ 2 , activation of lysophosphatidic acid receptors leads to the rapid and transient formation of a GRK/Gβγ complex. That Gβγ specificity exists at the level of the GPCR and the GRK is indicated by the observation that a GRK2/Gβγ complex is formed after agonist occupancy of the lysophosphatidic acid and β-adrenergic but not thrombin receptors. In contrast to GRK2, GRK3 forms a Gβγ complex after stimulation of all three GPCRs. This Gβγ binding specificity of the GRKs is also reflected at the level of the purified proteins. Thus the GRK2 carboxyl terminus binds Gβ 1 and Gβ 2 but not Gβ 3 , while the GRK3 fusion protein binds all three Gβ isoforms. This study provides a direct demonstration of a role for Gβγ in mediating the agonist-stimulated translocation of GRK2 and GRK3 in an intact cellular system and demonstrates isoform specificity in the interaction of these components.

Receptor-mediated activation of heterotrimeric guanine nucleotide-binding proteins (G proteins) results in the dissociation of alpha from beta gamma subunits, thereby allowing both to regulate effectors. Little is known about the regions of effectors required for recognition of G beta gamma. A peptide encoding residues 956 to 982 of adenylyl cyclase 2 specifically blocked G beta gamma stimulation of adenylyl cyclase 2, phospholipase C-beta 3, potassium channels, and beta-adrenergic receptor kinase as well as inhibition of calmodulin-stimulated adenylyl cyclases, but had no effect on interactions between G beta gamma and G alpha o. Substitutions in this peptide identified a functionally important motif, Gln-X-X-Glu-Arg, that is also conserved in regions of potassium channels and beta-adrenergic receptor kinases that participate in G beta gamma interactions. Thus, the region defined by residues 956 to 982 of adenylyl cyclase 2 may contain determinants important for receiving signals from G beta gamma.

Receptor-mediated activation of heterotrimeric guanine nucleotide-binding proteins (G proteins) results in the dissociation of α from βγ subunits, thereby allowing both to regulate effectors. Little is known about the regions of effectors required for recognition of Gβγ. A peptide encoding residues 956 to 982 of adenylyl cyclase 2 specifically blocked Gβγ stimulation of adenylyl cyclase 2, phospholipase C-β3, potassium channels, and β-adrenergic receptor kinase as well as inhibition of calmodulin-stimulated adenylyl cyclases, but had no effect on interactions between Gβγ and G$\\alpha_o$. Substitutions in this peptide identified a functionally important motif, Gln-X-X-Glu-Arg, that is also conserved in regions of potassium channels and β-adrenergic receptor kinases that participate in Gβγ interactions. Thus, the region defined by residues 956 to 982 of adenylyl cyclase 2 may contain determinants important for receiving signals from Gβγ.

The G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate and desensitize agonist-occupied GPCRs. GRK2-mediated receptor phosphorylation is preceded by the agonist-dependent membrane association of this enzyme. Previous in vitro studies with purified proteins have suggested that this translocation may be mediated by the recruitment of GRK2 to the plasma membrane by its interaction with the free β γ subunits of heterotrimeric G proteins (Gβ γ ). Here we demonstrate that this mechanism operates in intact cells and that specificity is imparted by the selective interaction of discrete pools of Gβ γ with receptors and GRKs. Treatment of Cos-7 cells transiently overexpressing GRK2 with a β -receptor agonist promotes a 3-fold increase in plasma membrane-associated GRK2. This translocation of GRK2 is inhibited by the carboxyl terminus of GRK2, a known Gβ γ sequestrant. Furthermore, in cells overexpressing both GRK2 and Gβ1γ2, activation of lysophosphatidic acid receptors leads to the rapid and transient formation of a GRK/Gβ γ complex. That Gβ γ specificity exists at the level of the GPCR and the GRK is indicated by the observation that a GRK2/Gβ γ complex is formed after agonist occupancy of the lysophosphatidic acid and β -adrenergic but not thrombin receptors. In contrast to GRK2, GRK3 forms a Gβ γ complex after stimulation of all three GPCRs. This Gβ γ binding specificity of the GRKs is also reflected at the level of the purified proteins. Thus the GRK2 carboxyl terminus binds Gβ1and Gβ2but not Gβ3, while the GRK3 fusion protein binds all three Gβ isoforms. This study provides a direct demonstration of a role for Gβ γ in mediating the agonist-stimulated translocation of GRK2 and GRK3 in an intact cellular system and demonstrates isoform specificity in the interaction of these components.

Daaka et al demonstrate a mechanism involving the G protein-coupled receptor (GPCR) kinases (GRKs) GRK2/GBetagamma and GRK3/GBetagamma. This complex formation mechanism in a cellular system reveals important insights into GBetagamma binding specificities of not only the GRKs but also specific GPCRs.

Stimulation of β 2 -adrenergic receptors on the cell surface by adrenaline or noradrenaline leads to alterations in the metabolism, excitability, differentiation and growth of many cell types. These effects have traditionally been thought to be mediated exclusively by receptor activation of intracellular G proteins 1 . However, certain physiological effects of β 2 -adrenergic receptor stimulation, notably the regulation of cellular pH by modulation of Na + /H + exchanger (NHE) function, do not seem to be entirely dependent on G-protein activation 2 , 3 , 4 , 5 , 6 , 7 . We report here a direct agonist-promoted association of the β 2 -adrenergic receptor with the Na + /H + exchanger regulatory factor (NHERF), a protein that regulates the activity of the Na + /H + exchanger type 3 (NHE3) 8 . NHERF binds to the β 2 -adrenergic receptor by means of a PDZ-domain-mediated interaction with the last few residues of the carboxy-terminal cytoplasmic domain of the receptor. Mutation ofthe final residue of the β 2 -adrenergic receptor from leucine toalanine abolishes the receptor's interaction with NHERF andalso markedly alters β 2 -adrenergic receptor regulation of NHE3 in cells without altering receptor-mediated activation of adenylyl cyclase. Our findings indicate that agonist-dependent β 2 -adrenergic receptor binding of NHERF plays a role in β 2 -adrenergic receptor-mediated regulation of Na + /H + exchange.

The role of the C-terminal helical thong in the stability of the E-coli glutamine synthetase dodecamer and the minimum glutamine synthetase structure needed for the regulatory adenylylation reaction were investigated. In the first part of this study, three site-directed mutants were created that truncate the helical thong of E-coli glutamine synthetase by 8, 3, or 1 amino acid(s) and were E461Stop, E463Stop, and V468Stop. The V468Stop mutant was dodecameric as seen from non-denaturing gel electrophoresis but the E463Stop and E461Stop mutants were mostly hexameric. There was a considerable amount of dodecamer present for E463Stop enzyme and the dodecamer for this mutant slowly dissociated to the hexameric form. The E461Stop hexamer and the E463Stop hexamer showed very little activity and neither hexamer could be adenylylated by adenylyltransferase (ATase). The E463Stop dodecamer, however, was active and could be adenylylated by ATase. The steady-state kinetic parameters showed that with increasing truncation there was a decrease in the specificity constant and k$\\sb{\\rm cat}$ for the dodecamers. The second part of the study addressed the question of the minimum quaternary structure of GS needed for the regulatory adenylylation reaction. To determine the amount of quaternary structure necessary for adenylylation, wild-type dodecamer (partially inactivated with 12% MSOX) was dissociated into a distribution of dodecamer, decamer, octamer, hexamer, and tetramer (GS Ladder) and used in the adenylylation reaction. Each of the GS ladder oligomers was adenylylated so the dodecameric structure was not essential for adenylylation. The adenylylation reaction was used next to determine the ratio of AMP bound per GS subunit for each of the oligomers. The ratios worked out to be 1.0 for the dodecamer, 0.75 for the octamer, 0.68 for the hexamer, and 0.47 for the tetramer after 6 hours of incubation. These ratios suggest that ATase needs the tetramer face of the dodecamer for adenylylation.

Metastasis is the primary cause of cancer-related deaths. Although The Cancer Genome Atlas has sequenced primary tumour types obtained from surgical resections, much less comprehensive molecular analysis is available from clinically acquired metastatic cancers. Here we perform whole-exome and -transcriptome sequencing of 500 adult patients with metastatic solid tumours of diverse lineage and biopsy site. The most prevalent genes somatically altered in metastatic cancer included TP53 , CDKN2A , PTEN , PIK3CA , and RB1 . Putative pathogenic germline variants were present in 12.2% of cases of which 75% were related to defects in DNA repair. RNA sequencing complemented DNA sequencing to identify gene fusions, pathway activation, and immune profiling. Our results show that integrative sequence analysis provides a clinically relevant, multi-dimensional view of the complex molecular landscape and microenvironment of metastatic cancers. Clinical exome and transcriptome sequencing of 500 adult patients with metastatic solid tumours of diverse lineage and biopsy site, as part of the Michigan Oncology Sequencing (MI-ONCOSEQ) Program. Genomic landscape of metastatic cancer Cancer cells often gain new mutations as they spread through the body from the primary tumour site and develop into metastatic tumours. Arul Chinnaiyan and colleagues report clinical whole exome and transcriptome sequencing of 500 adult patients with metastatic solid tumours of diverse lineages and biopsy sites, as part of the Michigan Oncology Sequencing (MI-ONCOSEQ) Program. The authors characterize the landscape of genomic alterations across metastatic cancers, including recurrent somatic alterations in TP53 , CDKN2A , PTEN , PIK3CA and RB1 . They also used clinical RNA sequencing to characterize gene fusions, transcriptional signatures and the immune microenvironment of metastatic cancer. A timely analysis of the genomic and molecular profiles of metastatic tumours could help to tailor anticancer therapies to patients more precisely than can profiling only primary tumours.

Participation in case-control studies is crucial in epidemiological research. The self-sampling bias, low response rate, and poor recruitment of population representative controls are often reported as limitations of case-control studies with limited strategies to improve participation. With greater use of web-based methods in health research, there is a further need to understand the effectiveness of different tools to enhance informed decision-making and willingness to take part in research. This study tests whether the inclusion of an animated decision aid in the recruitment page of a study website can increase participants' intentions to volunteer as controls. A total of 1425 women were included in a web-based experiment and randomized to one of two experimental conditions: one in which they were exposed to a simulated website that included the animation (animation; n=693, 48.6%), and one in which they were exposed to the simulated website without the animation (control; n=732, 51.4%). The simulated website was adapted from a real website for a case-control study, which invites people to consider taking part in a study that investigates differences in purchasing behaviors between women with and without ovarian cancer and share their loyalty card data collected through 2 high street retailers with the researchers. After exposure to the experimental manipulation, participants were asked to state (1) their intention to take part in the case-control study, (2) whether they would be willing to share their loyalty card for research, and (3) their willingness to be redirected to the real website after completing the survey. Data were assessed using ordinal and binary logistic regression, reported in percentages (%), adjusted odds ratio (AOR), and 95% confidence intervals. Including the animation in the simulated website did not increase intentions to participate in the study (AOR 1.09; 95% CI 0.88-1.35) or willingness to visit the real study website after the survey (control 50.5% vs animation 52.6%, AOR 1.08; 95% CI 0.85-1.37). The animation, however, increased the participants' intentions to share the data from their loyalty cards for research in general (control 17.9% vs animation 26%; AOR 1.64; 95% CI 1.23-2.18). While the results of this study indicate that the animated decision aid did not lead to greater intention to take part in our web-based case-control study, they show that they can be effective in increasing people's willingness to share sensitive data for health research.