In multicellular organisms from Caenorhabditis elegans to Homo sapiens, the maintenance of homeostasis is dependent on the continual flow and processing of information through a complex network of cells. Moreover, in order for the organism to respond to an ever-changing environment, intercellular signals must be transduced, amplified, and ultimately converted to the appropriate physiological response. The resolution of the molecular events underlying signal response and integration forms the basis of the signal transduction field of research. An evolutionarily highly conserved group of molecules known as heterotrimeric guanine nucleotide-binding proteins (G proteins) are key determinants of the specificity and temporal characteristics of many signaling processes and are the topic of this review. Numerous hormones, neurotransmitters, chemokines, local mediators, and sensory stimuli exert their effects on cells by binding to heptahelical membrane receptors coupled to heterotrimeric G proteins. These highly specialized transducers can modulate the activity of multiple signaling pathways leading to diverse biological responses. In vivo, specific combinations of G alpha- and G beta gamma-subunits are likely required for connecting individual receptors to signaling pathways. The structural determinants of receptor-G protein-effector specificity are not completely understood and, in addition to involving interaction domains of these primary acting proteins, also require the participation of scaffolding and regulatory proteins.

Heptahelical receptors activate intracellular signaling pathways by catalyzing GTP for GDP exchange on the heterotrimeric G protein alpha subunit (G alpha). Despite the crucial role of this process in cell signaling, little is known about the mechanism of G protein activation. Here we explore the structural basis for receptor-mediated GDP release using electron paramagnetic resonance spectroscopy. Binding to the activated receptor (R*) causes an apparent rigid-body movement of the alpha5 helix of G alpha that would perturb GDP binding at the beta6-alpha5 loop. This movement was not observed when a flexible loop was inserted between the alpha5 helix and the R*-binding C terminus, which uncouples R* binding from nucleotide exchange, suggesting that this movement is necessary for GDP release. These data provide the first direct observation of R*-mediated conformational changes in G proteins and define the structural basis for GDP release from G alpha.

The activation of G protein-coupled receptors (GPCRs) can result in an inhibition of Ca(2+)-dependent hormone and neurotransmitter secretion. This has been attributed in part to G protein inhibition of Ca(2+) influx. However, a frequently dominant inhibitory effect, of unknown mechanism, also occurs distal to Ca(2+) entry. Here we characterize direct inhibitory actions of G protein betagamma (Gbetagamma) on Ca(2+)-triggered vesicle exocytosis in permeable PC12 cells. Gbetagamma inhibition was rapid (<1 s) and was attenuated by cleavage of synaptosome-associated protein of 25 kD (SNAP25). Gbetagamma bound soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes, and binding was reduced to SNARE complexes containing cleaved SNAP25 or by Ca(2+)-dependent synaptotagmin binding. Here we show inhibitory coupling between GPCRs and vesicle exocytosis mediated directly by Gbetagamma interactions with the Ca(2+)-dependent fusion machinery.

In G-protein signaling, an activated receptor catalyzes GDP/GTP exchange on the G α subunit of a heterotrimeric G protein. In an initial step, receptor interaction with G α acts to allosterically trigger GDP release from a binding site located between the nucleotide binding domain and a helical domain, but the molecular mechanism is unknown. In this study, site-directed spin labeling and double electron-electron resonance spectroscopy are employed to reveal a large-scale separation of the domains that provides a direct pathway for nucleotide escape. Cross-linking studies show that the domain separation is required for receptor enhancement of nucleotide exchange rates. The interdomain opening is coupled to receptor binding via the C-terminal helix of G α , the extension of which is a high-affinity receptor binding element.signal transduction | structural polymorphism T he α-subunit (G α ) of heterotrimeric G proteins (G αβγ ) mediates signal transduction in a variety of cell signaling pathways (1). Multiple conformational states of G α are involved in the signal transduction pathway shown in Fig. 1A. In the inactive state, the G α subunit contains a bound GDP [G α ðGDPÞ] and has a high affinity for G βγ . When activated by an appropriate signal, a membrane-bound G-protein coupled receptor (GPCR) binds the heterotrimer in a quaternary complex, leading to the dissociation of GDP and formation of an "empty complex" [G α ð0Þ βγ ], which subsequently binds GTP. The affinity of G α ðGTPÞ for G βγ is dramatically reduced relative to G α ðGDPÞ, resulting in functional dissociation of active G α ðGTPÞ from the membrane-bound complex. The active G α ðGTPÞ subsequently binds downstream effector proteins to trigger a variety of regulatory events, depending on the particular system. Thus, the GPCR acts to catalyze GDP/GTP exchange via an empty complex. Crystallographic (2-7), biochemical (8), and biophysical (9-11) studies have elucidated details of the conformational states of G α that correspond to the discrete steps indicated in Fig. 1A, but the mechanism by which receptor interaction leads to release of the bound GDP from G α and the structure of the empty complex remain a major target of research in the field.The G α subunit has two structural domains, namely a nucleotide binding domain and a helical domain that partially occludes the bound nucleotide (Fig. 1B). From the initial G α crystal structure in 1993, Noel et al. (2) recognized that nucleotide release would probably require an opening between the two domains in the empty complex, but in the intervening 18 years there has been little compelling experimental support for this idea. Nevertheless, some constraints on the general topology of the complex are known. For example, numerous studies indicate that the C terminus of G α is bound tightly to the receptor in the empty complex (9). In addition, the N-terminal helix of G α is associated with G βγ and with the membrane via N-terminal myristoylation (12,13). Together, these constraints fix the position of the nucleot...

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