Receptor stimulation of nucleotide exchange in a heterotrimeric G protein (␣␤␥) is the primary event-modulating signaling by G proteins. The molecular mechanisms at the basis of this event and the role of the G protein subunits, especially the ␤␥ complex, in receptor activation are unclear. In a reconstituted system, a purified muscarinic receptor, M2, activates G protein heterotrimers ␣i2␤1␥5 and ␣i2␤1␥7 with equal efficacy. However, when the ␣ subunit type is substituted with ␣o, ␣o␤1␥7 shows a 100% increase in M2-stimulated GTP hydrolysis compared with ␣o␤1␥5. Using a sensitive assay based on ␤␥ complex stimulation of phospholipase C activity, we show that both ␤1␥5 and ␤1␥7 form heterotrimers equally well with ␣o and ␣i. These results indicate that the ␥ subunit interaction with a receptor is critical for modulating nucleotide exchange and is influenced by the subunit-type composition of the heterotrimer.The G protein cycle is primarily regulated by the interaction of heterotrimeric G protein and cell surface receptors. Both ␣ and ␤␥ subunits are required for interaction with receptor (1-3). The G protein ␣ subunit has been demonstrated to interact with and selectively couple to receptors, especially muscarinic receptors (4). The role of the ␤␥ complex in interaction with the receptor is, however, less well understood. There is evidence for interaction of the G protein ␥ subunit with receptors (5). There are also indications for specificity in this interaction. Results from experiments in pituitary GH3 cells using antisense oligonucleotides specific to different ␤ or ␥ subunit types indicated that signaling stimulated by different receptors can be specifically inhibited (6). The ␥1 subunit type allowed effective coupling of Gt with rhodopsin in contrast to ␥2 and ␥3 (7). In superior cervical ganglion (SCG) 1 neurons peptides specific to the ␥5 subunit type disrupted signaling from the M2/M4 muscarinic receptors, whereas peptides specific to ␥7 and ␥12 had no effect (8). Because this implied that the M2 muscarinic receptor selectively interacts with a G protein containing ␥5but not ␥7, we tested the ability of the M2 receptor to activate G proteins containing these two subunits. Earlier studies addressing the question of G protein specificity for receptors used whole cells or crude membranes from cells. Experiments with intact cells do not definitively allow identification of the site at which the disruption in signaling occurs. Crude membranes contain endogenous G proteins, receptors, and other components that may affect analysis of specificity in receptor-G protein interactions. To more rigorously and directly examine the effect of G protein subunit constitution on receptor-G protein coupling, we reconstituted a purified muscarinic receptor, M2, in lipids and measured its ability to activate G proteins containing different ␥ subunits. The M2 receptor is known to couple to members of the Gi/o family but not Gq (9). To examine whether the subunit-type constitution of a heterotrimer influenced receptor interac...

In comparison with the ␣ subunit of G proteins, the role of the ␤ subunit in signaling is less well understood. During the regulation of effectors by the ␤␥ complex, it is known that the ␤ subunit contacts effectors directly, whereas the role of the ␤ subunit is undefined in receptor-G protein interaction. Among the five G protein ␤ subunits known, the ␤ 4 subunit type is the least studied. We compared the ability of ␤␥ complexes containing ␤ 4 and the well characterized ␤ 1 to stimulate three different effectors: phospholipase C-␤2, phospholipase C-␤3, and adenylyl cyclase type II. ␤ 4 ␥ 2 and ␤ 1 ␥ 2 activated all three of these effectors with equal efficacy. However, nucleotide exchange in a G protein constituting ␣ o ␤ 4 ␥ 2 was stimulated significantly more by the M2 muscarinic receptor compared with ␣ o ␤ 1 ␥ 2 . Because ␣ o forms heterotrimers with ␤ 4 ␥ 2 and ␤ 1 ␥ 2 equally well, these results show that the ␤ subunit type plays a direct role in the receptor activation of a G protein.The G protein ␤␥ complex regulates the activity of a diverse set of effectors, including phospholipases, adenylyl cyclases, and ion channels (1). There is evidence that the ␤ subunit in the complex interacts directly with effectors (2-5). There are five ␤ subtypes (␤ 1 -␤ 5 ) as well as an alternatively spliced version of ␤ 5 (known as ␤ 5 -long) (6 -11). ␤ 1 -␤ 4 share over 80% identity with one another, whereas ␤ 5 shares only ϳ50% identity with the other ␤ subunits (12). The divergence between ␤ 5 and the other ␤ subunits is consistent with the functional differences between ␤ 5 and ␤ 1 observed in effector regulation in a variety of systems (4,13,14). The high sequence similarity of ␤ 1 -␤ 4 suggests that their functions are conserved. Although some experiments indicated little difference in effector modulating capability among these ␤ subunit types, other experiments suggest otherwise. The G protein-coupled receptor kinase GRK3 binds ␤␥ complexes consisting of ␤ 1 , ␤ 2 , and ␤ 3 , but only ␤ 1 and ␤ 2 bind to the related kinase GRK2 (15). Other results indicate the selective mediation of cross-talk between G proteins and protein kinase C modulation of N-type channels by the ␤ 1 subunit type (16).Experiments focusing on the specific role of individual G protein subunit types have provided evidence for a certain level of selectivity in the interaction of ␣ subunit types with receptors (17). Evidence for similar selectivity of interaction between ␥ subunit types and receptors also exists (18 -20). In contrast there is limited evidence for ␤ subunit type selectivity in receptor interaction. Whole-cell experiments using antisense oligonucleotides directed against specific ␤ subunit cDNAs selectively disrupted signaling from particular receptors (21). Although the selective interaction of ␤ subunit types with receptors could give rise to this result, such selectivity has not been shown so far.Among the five ␤ subunits, ␤ 4 is the least studied. Its role in effector regulation and receptor interaction remains unclear. To exam...

Screening assays using target-based affinity selection coupled with high-sensitivity detection technologies to identify smallmolecule hits from chemical libraries can provide a useful discovery approach that complements traditional assay systems. Affinity selection-mass spectrometry (AS-MS) is one such methodology that holds promise for providing selective and sensitive high-throughput screening platforms. Although AS-MS screening platforms have been used to discover smallmolecule ligands of proteins from many target families, they have not yet been used routinely to screen integral membrane proteins. The authors present a proof-of-concept study using size exclusion chromatography coupled to AS-MS to perform a primary screen for small-molecule ligands of the purified muscarinic M 2 acetylcholine receptor, a G-protein-coupled receptor. AS-MS is used to characterize the binding mechanisms of 2 newly discovered ligands. NGD-3350 is a novel M 2 -specific orthosteric antagonist of M 2 function. NGD-3366 is an allosteric ligand with binding properties similar to the allosteric antagonist W-84, which decreases the dissociation rate of N-methyl-scopolamine from the M 2 receptor. Binding properties of the ligands discerned from AS-MS assays agree with those from in vitro biochemical assays. The authors conclude that when used with appropriate small-molecule libraries, AS-MS may provide a useful high-throughput assay system for the discovery and characterization of all classes of integral membrane protein ligands, including allosteric modulators. (Journal of Biomolecular

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