Gβ5 prevents the RGS7-Gαo interaction through binding to a distinct Gγ-like domain found in RGS7 and other RGS proteins

The G protein β subunit Gβ5 deviates significantly from the other four members of Gβ-subunit family in amino acid sequence and subcellular localization. To detect the protein targets of Gβ5 in vivo, we have isolated a native Gβ5 protein complex from the retinal cytosolic fraction and identified the protein tightly associated with Gβ5 as the regulator of G protein signaling (RGS) protein, RGS7. Here we show that complexes of Gβ5 with RGS proteins can be formed in vitro from the recombinant proteins. The reconstituted Gβ5-RGS dimers are similar to the native retinal complex in their behavior on gel-filtration and cation-exchange chromatographies and can be immunoprecipitated with either anti-Gβ5 or anti-RGS7 antibodies. The specific Gβ5-RGS7 interaction is determined by a distinct domain in RGS that has a striking homology to Gγ subunits. Deletion of this domain prevents the RGS7-Gβ5 binding, although the interaction with Gα is retained. Substitution of the Gγ-like domain of RGS7 with a portion of Gγ1 changes its binding specificity from Gβ5 to Gβ1. The interaction of Gβ5 with RGS7 blocked the binding of RGS7 to the Gα subunit Gαo, indicating that Gβ5 is a specific RGS inhibitor

Identification of the Gβ5-RGS7 Protein Complex in the Retina

The G protein β subunit Gβ5 deviates significantly from the four other members of the Gβ family in amino acid sequence, unique expression pattern (only in the CNS), and cytosolic localization. To identify the members of the Gβ5-mediated signaling pathway, we purified the native protein complex containing Gβ5 from the cytosolic fraction of bovine retina. Analysis of the isolated complex revealed that Gβ5 is tightly associated with RGS7, a member of the superfamily of negative regulators of G protein signaling. This finding, for the first time, demonstrates an interaction between a Gβ subunit and an RGS protein. Gβ5 was not detected in the outer segments of photoreceptor cells, suggesting that the cytosolic Gβ5-RGS7 complex is not directly involved in phototransduction

Localization of the Sites for Ca 2+ -Binding Proteins on G Protein-Coupled Receptor

Inhibition of G protein-coupled receptor kinases (GRKs) by Ca 2+ -binding proteins has recently emerged as a general mechanism of GRK regulation. While GRK1 (rhodopsin kinase) is inhibited by the photoreceptor-specific Ca 2+ -binding protein recoverin, other GRKs can be inhibited by Ca 2+ -calmodulin. To dissect the mechanism of this inhibition at the molecular level, we localized the GRK domains involved in Ca 2+ -binding protein interaction using a series of GST-GRK fusion proteins. GRK1, GRK2, and GRK5, which represent the three known GRK subclasses, were each found to possess two distinct calmodulin-binding sites. These sites were localized to the N- and C-terminal regulatory regions within domains rich in positively charged and hydrophobic residues. In contrast, the unique N-terminally localized GRK1 site for recoverin had no clearly defined structural characteristics. Interestingly, while the recoverin and calmodulin-binding sites in GRK1 do not overlap, recoverin-GRK1 interaction is inhibited by calmodulin, most likely via an allosteric mechanism. Further analysis of the individual calmodulin sites in GRK5 suggests that the C-terminal site plays the major role in GRK5-calmodulin interaction. While specific mutation within the N-terminal site had no effect on calmodulin-mediated inhibition of GRK5 activity, deletion of the C-terminal site attenuated the effect of calmodulin on GRK5, and the simultaneous mutation of both sites rendered the enzyme calmodulin-insensitive. These studies provide new insight into the mechanism of Ca 2+ -dependent regulation of GRKs

Subunit contributions to phosphorylation-dependent modulation of bovine rod cyclic nucleotide-gated channels

Cyclic nucleotide-gated (CNG) channels in rod photoreceptors transduce a decrease in cGMP into hyperpolarization during the light response. Insulin-like growth factor-1 (IGF-1) increases light responses by increasing the cGMP sensitivity of CNG channels, an event mediated by a protein tyrosine phosphatase. Native rod CNG channels are heteromultimers, composed of three CNGA1 subunits and one CNGB1 subunit. Previous studies on heterologously expressed rod CNG channels show that a specific tyrosine in the CNGA1 subunit (Y498) is required for modulation by protein tyrosine phosphatases, protein tyrosine kinases and IGF-1. Here we show that the CNGB1 subunit contains a specific tyrosine (Y1097) that is important for modulation of heteromeric channels by tyrosine phosphorylation. Direct biochemical measurements demonstrate 32P-labelling of CNGA1Y498 and CNGB1Y1097. Replacement of either Y498 of CNGA1 or Y1097 of CNGB1 with phenylalanine reduces modulation, and removal of both tyrosines eliminates modulation. Unlike CNGA1, CNGB1 does not exhibit activity dependence of modulation by tyrosine phosphorylation. Hence both CNGA1 and CNGB1 subunits contribute to phosphorylation-dependent modulation of rod CNG channels, but the phosphorylation states of the two subunits are regulated in different ways

Localization of the Sites for Ca2+-Binding Proteins on G Protein-Coupled Receptor Kinases

Inhibition of G protein-coupled receptor kinases (GRKs) by Ca2+-binding proteins has recently emerged as a general mechanism of GRK regulation. While GRK1 (rhodopsin kinase) is inhibited by the photoreceptor-specific Ca2+-binding protein recoverin, other GRKs can be inhibited by Ca2+−calmodulin. To dissect the mechanism of this inhibition at the molecular level, we localized the GRK domains involved in Ca2+-binding protein interaction using a series of GST−GRK fusion proteins. GRK1, GRK2, and GRK5, which represent the three known GRK subclasses, were each found to possess two distinct calmodulin-binding sites. These sites were localized to the N- and C-terminal regulatory regions within domains rich in positively charged and hydrophobic residues. In contrast, the unique N-terminally localized GRK1 site for recoverin had no clearly defined structural characteristics. Interestingly, while the recoverin and calmodulin-binding sites in GRK1 do not overlap, recoverin−GRK1 interaction is inhibited by calmodulin, most likely via an allosteric mechanism. Further analysis of the individual calmodulin sites in GRK5 suggests that the C-terminal site plays the major role in GRK5−calmodulin interaction. While specific mutation within the N-terminal site had no effect on calmodulin-mediated inhibition of GRK5 activity, deletion of the C-terminal site attenuated the effect of calmodulin on GRK5, and the simultaneous mutation of both sites rendered the enzyme calmodulin-insensitive. These studies provide new insight into the mechanism of Ca2+-dependent regulation of GRKs

Autophosphorylation and ADP Regulate the Ca^(2+)-Dependent Interaction of Recoverin with Rhodopsin Kinase

Recoverin is a 23 kDa myristoylated Ca^(2+)-binding protein that inhibits rhodopsin kinase. We have used surface plasmon resonance to investigate the influences of Ca^(2+), myristoylation, and adenine nucleotides on the recoverin−rhodopsin kinase interaction. Our analyses confirmed that Ca^(2+) is required for recoverin to bind RK. Myristoylation had little effect on the affinity of recoverin for the kinase, but it raised the K_(0.5) for Ca^(2+) from 150 nM for nonacylated recoverin to 400 nM for myristoylated recoverin. Finally, our studies also revealed two separate and previously unreported effects of adenine nucleotides on the recoverin−rhodopsin kinase binding. The interaction is weakened by autophosphorylation of the kinase, and it is strengthened by the presence of ADP

Autophosphorylation and ADP Regulate the Ca^(2+)-Dependent Interaction of Recoverin with Rhodopsin Kinase

Recoverin is a 23 kDa myristoylated Ca^(2+)-binding protein that inhibits rhodopsin kinase. We have used surface plasmon resonance to investigate the influences of Ca^(2+), myristoylation, and adenine nucleotides on the recoverin−rhodopsin kinase interaction. Our analyses confirmed that Ca^(2+) is required for recoverin to bind RK. Myristoylation had little effect on the affinity of recoverin for the kinase, but it raised the K_(0.5) for Ca^(2+) from 150 nM for nonacylated recoverin to 400 nM for myristoylated recoverin. Finally, our studies also revealed two separate and previously unreported effects of adenine nucleotides on the recoverin−rhodopsin kinase binding. The interaction is weakened by autophosphorylation of the kinase, and it is strengthened by the presence of ADP

A global benchmark study using affinity-based biosensors

International audienceTo explore the variability in biosensor studies, 150 participants from 20 countries were given the same protein samples and asked to determine kinetic rate constants for the interaction. We chose a protein system that was amenable to analysis using different biosensor platforms as well as by users of different expertise levels. The two proteins (a 50-kDa Fab and a 60-kDa glutathione S-transferase [GST] antigen) form a relatively high-affinity complex, so participants needed to optimize several experimental parameters, including ligand immobilization and regeneration conditions as well as analyte concentrations and injection/dissociation times. Although most participants collected binding responses that could be fit to yield kinetic parameters, the quality of a few data sets could have been improved by optimizing the assay design. Once these outliers were removed, the average reported affinity across the remaining panel of participants was 620 pM with a standard deviation of 980 pM. These results demonstrate that when this biosensor assay was designed and executed appropriately, the reported rate constants were consistent, and independent of which protein was immobilized and which biosensor was used. (C) 2008 Elsevier Inc. All rights reserved