The recognition between G protein and cognate receptor plays a key role in specific cellular responses to environmental stimuli. Here we explore specificity in receptor-G protein coupling by taking advantage of the ability of the 5-hydroxytryptamine 1B (5-HT 1B ) receptor to discriminate between G protein heterotrimers containing G␣ i1 or G␣ t . G i1 can interact with the 5-HT 1B receptor and stabilize a high affinity agonist binding state of this receptor, but G t cannot. A series of G␣ t /G␣ i1 chimeric proteins have been generated in Escherichia coli, and their functional integrity has been reported previously (Skiba, N. P., Bae, H., and Hamm, H. E. (1996) J. Biol. Chem. 271, 413-424). We have tested the functional coupling abilities of the G␣ t /G␣ i1 chimeras to 5-HT 1B receptors using high affinity agonist binding and receptor-stimulated guanosine 5-3-O-(thio)triphosphate (GTP␥S) binding. In the presence of ␤␥ subunits, amino acid residues 299 -318 of G␣ i1 increase agonist binding to the 5-HT 1B receptor and receptor stimulation of GTP␥S binding. Moreover, G␣ i1 containing only G␣ t amino acid sequences from this region does not show any coupling ability to 5-HT 1B receptors. Our studies suggest that the ␣4 helix and ␣4-␤6 loop region of G␣s are an important region for specific recognition between receptors and G i family members.The heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) mediate signaling from a large number of diverse heptahelical cell surface receptors to a variety of intracellular effectors. These pathways control numerous essential functions in all tissues and are ubiquitous throughout eukaryotes (1-3). A large body of work investigating the mechanisms underlying receptor-G protein interactions now exists. The early view that signaling selectivity would manifest itself on the basis of specific protein interactions allowing a receptor to couple with a unique G protein to modulate a single effector is no longer tenable with the accumulating evidence of a network of interactions that converge and diverge at multiple levels. Even in the earliest receptor-G protein reconstitution studies using phospholipid vesicles, it was clear that, while there were large differences in the efficiencies of coupling among the major families of G proteins, receptors were capable of activating multiple G proteins from distinct families (4, 5).Elucidation of the crystal structures of ␣ subunits in both active (6, 7) and inactive conformations (8), an isolated ␤␥ subunit (9) and the ␣␤␥ heterotrimer (10, 11), has begun to define a mechanistic basis for data from mutagenesis, chimera, and peptide studies defining functional domains on G protein subunits (12)(13)(14)(15)(16)(17). A variety of studies have implicated the C terminus of ␣ subunits in mediating receptor-G protein selectivity (13-15). Synthetic peptides from the C terminus of ␣ t (amino acids 340 -350) have been shown to stabilize the active conformation of metarhodopsin II (17) while alanine scanning mutagenesis of the same regi...

The molecular basis of selectivity in G-protein receptor coupling has been explored by comparing the abilities of G-protein heterotrimers containing chimeric G␣ subunits, comprised of various regions of G i1 ␣, G t ␣, and G q ␣, to stabilize the high affinity agonist binding state of serotonin, adenosine, and muscarinic receptors. The data indicate that multiple and distinct determinants of selectivity exist for individual receptors. While the A1 adenosine receptor does not distinguish between G i1 ␣ and G t ␣ sequences, the 5-HT 1A and 5-HT 1B serotonin and M2 muscarinic receptors can couple with G i1 but not G t . It is possible to distinguish domains that eliminate coupling and are defined as "critical," from those that impair coupling and are defined as "important." Domains within the N terminus, ␣4-helix, and ␣4-helix-␣4/␤6-loop of G i1 ␣ are involved in 5-HT and M2 receptor interactions. Chimeric G i1 ␣/G q ␣ subunits verify the critical role of the G␣ C terminus in receptor coupling, however, the individual receptors differ in the C-terminal amino acids required for coupling. Furthermore, the EC 50 for interactions with G i1 differ among the individual receptors. These results suggest that coupling selectivity ultimately involves subtle and cooperative interactions among various domains on both the G-protein and the associated receptor as well as the G-protein concentration.A large number of diverse seven transmembrane-spanning cell surface receptors mediate signaling to a variety of intracellular effectors by coupling to the heterotrimeric guanine nucleotide-binding regulatory proteins (G-proteins) 1 (1). The mechanisms responsible for selectivity in G-protein-mediated signaling pathways are not fully understood (2, 3). Although it is known that at the molecular level the selectivity in G-protein receptor coupling is determined by amino acid sequences of both receptor and G-protein, the individual amino acids involved in this selective recognition have not been completely identified. Different receptor systems and different methodologies indicate that the G␣ subunit C terminus and ␣5-helix (4 -7), N terminus, and ␣N-helix (4, 8 -10), ␣4-helix, and ␣4/ ␤6-loop (11-13), ␣2-helix, and ␣2/␤4-loop (14), ␣3/␤5-loop (15), ␣N/␤1-loop (13) and amino acids 110 -119 from the ␣-helical domain (16) are involved in receptor-coupling selectivity. Some of these domains contact the receptor directly, while others regulate receptor-coupling selectivity indirectly by playing a role in nucleotide exchange. Despite the fact that many of the receptor-interacting domains have been identified, the relationship between receptor subtypes and G␣ domains involved in receptor coupling has not been clearly established. Thus, it is difficult to predict which G␣ domains will be utilized by a specific receptor. Here we propose that individual receptors recognize specific patterns formed by amino acids of G␣ thus making G-protein interface look different for different receptors. The C terminus of G␣ is a well accepted receptor recognit...

The D 2 dopamine receptor has been expressed in Sf21 insect cells together with the G proteins G o and G i2 , using the baculovirus system. Expression levels of receptor and G protein (␣, ␤, and ␥ subunits) in the two preparations were similar as shown by binding of There is considerable interest in understanding the action mechanisms of agonists at receptors (1-3). Agonists must bind to receptors, and this may be characterized in terms of an affinity of agonist binding. Agonists must also activate the receptor and associated signaling systems, and this property is often referred to as efficacy. Efficacy is exhibited in terms of the maximal effect induced by the agonist and also in the EC 50 of the agonist in activating the signaling system, which is often lower than the concentration of agonist which achieves halfmaximal occupancy of the receptor.For G protein-coupled receptors, an influential model of agonist action is the ternary complex model and its recent extensions (4 -6). In this model the receptor exists in an inactive ground state, which may isomerize to a partially activated state (R*) 1 that is able to couple more efficiently to the G protein to form the coupled active species (R*G). The formation of R*G may occur spontaneously, but in the presence of an agonist both R* and R*G are stabilized, and the ternary complex (AR*G) is formed. Guanine nucleotide exchange (GDP/ GTP) occurs in both the binary complex (R*G) and the ternary complex (AR*G). The binary and ternary complexes dissociate releasing ␣GTP and ␤␥ subunits of the G protein which can alter effector activity. The agonist may also influence ternary complex breakdown (7,8) so that there are several places at which agonism is determined.There is, however, evidence that some receptors may interact with more than one G protein so that influences on different signaling pathways can occur. If a receptor can interact with more than one G protein this may influence the potency of agonist action and the pattern of agonist effects, i.e. the pharmacological profile of the response observed through the different G proteins. For the 5HT1A serotonin receptor, it was shown that the receptor interacts preferentially with G i /G o /G z subtypes of G protein (9) and that the nature of the G protein subtype influenced the agonist selectivity of the response (10). This question was addressed more explicitly for the ␣ 2 -adrenergic receptor (11). Expression of G␣ o , together with the endogenous G proteins of NIH 3T3 cells, altered the agonist selectivity of the receptor; the partial agonists, oxymetazoline and clonidine, exhibited increased efficacy. The possibility that the pharmacological profile of the response depends on the nature of the G protein has been termed "agonist trafficking" (12).The D 2 dopamine receptor has been shown to interact with different G proteins to influence different signaling events (13,14). In one study, interaction with G o has been shown to lead to inhibition of calcium channels, whereas interaction with G i subtypes has been show...

Near-infrared (NIR) spectroscopy has gained wide acceptance within the food and agriculture industries as a rapid analytical tool. NIR spectroscopy offers the advantage of rapid, non-destructive analysis and routine operation is simple and opens the possibility of using spectra to obtain the 'fingerprint' of a sample. The aim of this study was to explore the potential of combining visible (VIS) and nearinfrared (NIR) spectroscopy, together with multivariate analysis, in establishing the function of genes, by investigating the metabolic profiles produced by Saccharomyces cerevisiae deletion strains sourced from the EUROSCARF yeast collection. Spectra (400-2500 nm) were acquired with a FOSS NIRSystems6500 (Foss NIRSystems), in transmittance mode. Principal component analysis (PCA) and linear discriminant analysis (LDA) were used in order to visualize graphically the relative differences and similarities of yeast deletion strains. VIS and NIR spectroscopy showed great promise as a screening tool for both discriminating between yeast strains and grouping strains with deletions in genes that disturb similar metabolic pathways. These results indicate that the methods may be useful in defining the function of genes that produce no obvious phenotype.

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