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The interdomain interaction is identified, highlighting the advantage of modular molecules such as R7 family of regulators of G protein signaling (RGSs). The findings reveal that multidomain architecture permits coordination and acceleration of signaling events.

A study was conducted to identify the dynamic intramolecular interaction between the G[[beta].sub.5] and DEP domains in the G protein signaling (RGS) required for stabilizing and binding of DEP-G[[beta].sub.5] and their effects of on the interaction of G[[beta].sub.5]-RGS7 with the M3R. The data was found to be consistent with the model interaction of the M3R with G[[beta].sub.5]- RGS7 which caused the DEP domain and G[[beta].sub.5] to dissociate from each other and bind to the C-tail, and the binding of DEP domain to the third loop, which inhibited M3R-mediated signaling.

The ability of the G[[beta].sub.5]-RGS7 complex (RGS = Regulators of G protein signaling) to selectively inhibit [Ca.sup.2+] mobilization by muscarinic acetylcholine receptor type 3 (M3R), but not by other Gq-coupled receptors such as M1, M5, histamine H1, and GNRH receptors is demonstrated. The study identified a novel molecular mechanism that could impart receptor subtype selectivity on signal transduction via Gq-coupled muscarinic receptors.

The complex of the regulator of G protein signaling (RGS), Gβ5-RGS7, can inhibit signal transduction via the M3 muscarinic acetylcholine receptor (M3R). RGS7 Consists of three distinct structural entities: the DEP domain and its extension DHEX, the Gγ-like (GGL) domain, which is permanently bound to Gβ subunit Gβ5, and the RGS domain responsible for the interaction with Gα subunits. Inhibition of the M3R by Gβ5-RGS7 is independent of the RGS domain but requires binding of the DEP domain to the third intracellular loop of the receptor. Recent studies identified the dynamic intramolecular interaction between the Gβ5 and DEP domains, which suggested that the Gβ5-RGS7 dimer could alternate between the "open" and "closed" conformations. Here, we identified point mutations that weaken DEP-Gβ5 binding, presumably stabilizing the open state, and tested their effects on the interaction of Gβ5 -RGS7 with the M3R. We found that these mutations facilitated binding ofGβ5-RGS7 to the recombinant third intracellular loop of the M3R but did not enhance its ability to inhibit M3R-mediated Ca2+ mobilization. This led us to the idea that the M3R can effectively induce the Gβ5 -RGS7 dimer to open; such a mechanism would require a region of the receptor distinct from the third loop. Indeed, we found that the C-terminus of M3R interacts with Gβ5-RGS7. Truncation of the C-terminus rendered the M3R insensitive to inhibition by wild-type Gβ5 -RGS7; however, the open mutant of Gβ5 -RGS7 was able to inhibit signaling by the truncated M3R. The GST fusion of the M3R C-tail could not bind to wild-type Gβ5 -RGS7 but could associate with its open mutant as well as with the separated recombinant DEP domain or Gβ5 . Taken together, our data are consistent with the following model: interaction of the M3R with Gβ5-RGS7 causes the DEP domain and Gβ5 to dissociate from each other and bind to the C-tail, and the DEP domain also binds to the third loop, thereby inhibiting M3R-mediated signaling. [ABSTRACT FROM AUTHOR]

Regulators of G protein signaling (RGS) make up a diverse family primarily known as GTPase-activating proteins (GAPs) for heterotrimeric G proteins. In addition to the RGS domain, which is responsible for GAP activity, most RGS proteins contain other distinct structural motifs. For example, members of the R7 family of RGS proteins contain a DEP, GGL, and novel DHEX domain and are obligatory dimers with G protein β subunit Gβ5. Here we show that the Gβ5-RGS7 complex can inhibit Ca2+ mobilization elicited by muscarinic acetylcholine receptor type 3 (M3R), but not by other Gq-coupled receptors such as M1, M5, histamine H1, and GNRH receptors. The isolated DEP domain of RGS7 is sufficient for the inhibition of M3R signaling, whereas the deletion of the DEP domain renders the Gβ5-RGS7 complex ineffective. Deletion of a portion of the third intracellular loop allowed the receptor (M3R-short) to signal but rendered it insensitive to the effect of the Gβ5-RGS7 complex. Accordingly, the recombinant DEP domain bound in vitro to the GST-fused i3 loop of the M3R. These results identify a novel molecular mechanism that can impart receptor subtype selectivity on signal transduction via Gq-coupled muscarinic receptors. [ABSTRACT FROM AUTHOR]

The R7 family of RGS proteins (RGS6, -7, -9, -11) is characterized by the presence of three domains: DEP, GGL, and RGS. The RGS domain interacts with Gα subunits and exhibits GAP activity. The GGL domain permanently associates with Gβ5. The DEP domain interacts with the membrane anchoring protein, R7BP. Here we provide evidence for a novel interaction within this complex: between the DEP domain and Gβ5. GST fusion of the RGS7 DEP domain (GST-R7DEP) binds to both native and recombinant Gβ5-RGS7, recombinant Gβγ complexes, and monomeric Gβ5 and Gβ1 subunits. Co-immunoprecipitation and FRET assays supported the GST pull-down experiments. GST-R7DEP reduced FRET between CFP-Gβ5 and YFP-RGS7, indicating that the DEP-Gβ5 interaction is dynamic. In transfected cells, R7BP had no effect on the Gβ5/RGS7 pull down by GST-R7DEP. The DEP domain of RGS9 did not bind to Gβ5. Substitution of RGS7 Glu-73 and Asp-74 for the corresponding Ser and Gly residues (ED/SG mutation) of RGS9 diminished the DEP-Gβ5 interaction. In the absence of R7BP both the wild-type RGS7 and the ED/SG mutant attenuated muscarinic M3 receptor-mediated Ca2+ mobilization. En the presence of R7BP, wild-type RGS7 lost this inhibitory activity, whereas the ED/SG mutant remained active. Taken together, our results are consistent with the following model. The Gβ5-RGS7 molecule can exist in two conformations: ‘closed’ and ‘open’, when the DEP domain and Gβ5 subunit either do or do not interact. The closed conformation appears to be less active with respect to its effect on Gq-mediated signaling than the open conformation. [ABSTRACT FROM AUTHOR]