The crystallographic structure of the G protein heterotrimer Gi alpha 1(GDP)beta 1 gamma 2 (at 2.3 A) reveals two nonoverlapping regions of contact between alpha and beta, an extended interface between beta and nearly all of gamma, and limited interaction of alpha with gamma. The major alpha/beta interface covers switch II of alpha, and GTP-induced rearrangement of switch II causes subunit dissociation during signaling. Alterations in GDP binding in the heterotrimer (compared with alpha-GDP) explain stabilization of the inactive conformation of alpha by beta gamma. Repeated WD motifs in beta form a circularized sevenfold beta propeller. The conserved cores of these motifs are a scaffold for display of their more variable linkers on the exterior face of each propeller blade.

Acetylcholine released during parasympathetic stimulation of the vagal nerve slows the heart rate through the activation of muscarinic receptors and subsequent opening of an inwardly rectifying potassium channel. The activation of these muscarinic potassium channels is mediated by a pertussis toxin-sensitive heterotrimeric GTP-binding protein (G protein). It has not been resolved whether exogenously applied G alpha or G beta gamma, or both, activate the channel. Using a heterologous expression system, we have tested the ability of different G protein subunits to activate the cloned muscarinic potassium channel, GIRK1. We report here that coexpression of GIRK1 with G beta gamma but not G alpha beta gamma in Xenopus oocytes results in channel activity that persists in the absence of cytoplasmic GTP. This activity is reduced by fusion proteins of the beta-adrenergic receptor kinase and of recombinant G alpha i-GDP, both of which are known to interact with G beta gamma. Moreover, application of recombinant G beta gamma, but not G alpha i-GTP-gamma S, activates GIRK1 channels. Thus G beta gamma appears to be sufficient for the activation of GIRK1 muscarinic potassium channels.

Acetylcholine activates inwardly rectifying potassium channels (IK.ACh) in the heart through muscarinic receptor binding and activation of pertussis-toxin-sensitive G proteins. Experiments showing that only the beta gamma-subunit (G beta gamma) activates IK.ACh (ref. 4) were challenged by reports that only the activated alpha-subunit (G alpha) was effective. Here we examine IK.ACh regulation using purified brain and recombinant G-protein subunits. Six recombinant G beta gamma-subunits activated IK.ACh with apparent half-maximal activation concentrations of 3-30 nM. Activation of IK.ACh by recombinant G alpha-GTP gamma S was observed, but this was probably due to release of GTP gamma S from the protein. Importantly, IK.ACh activity elicited by GTP gamma S was inhibited by purified brain and recombinant G alpha-GDP, suggesting that native G beta gamma plays a major role in this pathway. We conclude that G beta gamma is a primary regulator of IK.ACh activity.

Evidence suggests that both alpha and beta gamma subunits of heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) inhibit adenylyl cyclase. Although type I adenylyl cyclase is inhibited directly by exogenous beta gamma, inhibition of adenylyl cyclase by Gi alpha has not been convincingly demonstrated in vitro. Concentration-dependent inhibition of adenylyl cyclases by purified Gi alpha subunits is described. Activated Gi alpha but not G(o) alpha was effective, and myristoylation of Gi alpha was required. The characteristics of the inhibitory effect were dependent on the type of adenylyl cyclase and the nature of the activator of the enzyme. The concentrations of Gi alpha required to inhibit adenylyl cyclase were substantially higher than those normally thought to be relevant physiologically. However, analysis indicates that these concentrations may be relevant and reasonable.

DNA regulatory elements frequently harbor multiple recognition sites for several transcriptional activators. The response mounted from such compound response elements is often more pronounced than the simple sum of effects observed at single binding sites. The determinants of such transcriptional synergy and its control, however, are poorly understood. Through a genetic approach, we have uncovered a novel protein motif that limits the transcriptional synergy of multiple DNA-binding regulators. Disruption of these conserved synergy control motifs (SC motifs) selectively increases activity at compound, but not single, response elements. Although isolated SC motifs do not regulate transcription when tethered to DNA, their transfer to an activator lacking them is sufficient to impose limits on synergy. Mechanistic analysis of the two SC motifs found in the glucocorticoid receptor N-terminal region reveals that they function irrespective of the arrangement of the receptor binding sites or their distance from the transcription start site. Proper function, however, requires the receptor's ligand-binding domain and an engaged dimer interface. Notably, the motifs are not functional in yeast and do not alter the effect of p160 coactivators, suggesting that they require other nonconserved components to operate. Many activators across multiple classes harbor seemingly unrelated negative regulatory regions. The presence of SC motifs within them, however, suggests a common function and identifies SC motifs as critical elements of a general mechanism to modulate higher-order interactions among transcriptional regulators.The development and physiology of higher eukaryotes rely on the accurate transcription of a large array of independent genes in response to specific temporal, spatial, and physiological cues. The information to establish such a complex program of gene expression is ultimately encoded in the regulatory DNA elements of each gene. Invariably, such cis-regulatory elements consist of clusters of recognition sites for various factors, often in multiple copies or partially overlapping each other (2). These units nucleate the cooperative assembly of multiprotein complexes or enhanceosomes (4). The final transcriptional output, however, is not the simple arithmetic addition of the independent effects of individual regulators. On the contrary, the integrated response of the gene is the result of a complex set of logical and quantitative operations that rely on the combinatorial coordination of multiple regulatory sites, factors, and signals (56-58).A central element of this regulatory logic that serves as both an amplification and specificity mechanism is the more-thanadditive, i.e., synergistic, response resulting from the recruitment of a given activator to multiple copies of a recognition site. This general form of interaction is observed even with artificial activators and thus may be an intrinsic consequence of the combinatorial design of eukaryotic transcription systems (25). The mechanisms and determinants that en...

Lysophosphatidic acid receptors stimulate a GA 12/13

The hormone-activated glucocorticoid receptor (GR), through its N-and C-terminal transcriptional activation functions AF-1 and AF-2, controls the transcription of target genes presumably through interaction(s) with transcriptional regulatory factors. Utilizing a modified yeast two-hybrid approach, we have identified the tumor susceptibility gene 101 (TSG101) and the vitamin D receptor-interacting protein 150 (DRIP150) as proteins that interact specifically with a functional GR AF-1 surface. In yeast and mammalian cells, TSG101 represses whereas DRIP150 enhances GR AF-1-mediated transactivation. Thus, GR AF-1 is capable of recruiting both positive and negative regulatory factors that differentially regulate GR transcriptional enhancement. In addition, we show that another member of the DRIP complex, DRIP205, interacts with the GR ligand binding domain in a hormone-dependent manner and facilitates GR transactivation in concert with DRIP150. These results suggest that DRIP150 and DRIP205 functionally link GR AF-1 and AF-2, and represent important mediators of GR transcriptional enhancement.

Functional interactions between factors bound at multiple sites on DNA often lead to a synergistic or more-than-additive transcriptional response. We previously defined a class of peptide sequences termed synergy control motifs (SC motifs) that function in multiple regulators by selectively inhibiting synergistic activity driven from multiple but not single response elements. By studying the prototypic SC motifs of the glucocorticoid receptor, we show that SC motifs inhibit transcription per se both in cis and in trans, and that a requirement for multiple contacts with DNA renders them selective for compound response elements. Notably, SC motifs are sites for SUMOylation, and the degree of modification correlates strongly with the extent of synergy control. Recruiting SUMO to the promoter either independently or as a fusion to the glucocorticoid receptor is sufficient to recapitulate the in trans and in cis inhibition by SC motifs without apparent changes in subcellular localization. Moreover, we find that the core ubiquitin fold domain of SUMO is sufficient for inhibition and that, independently of their potential for polySUMO chain formation, SUMO-2 and SUMO-3 are more effective inhibitors than SUMO-1. E ukaryotic transcriptional control is highly combinatorial, and the mosaic of response elements in the regulatory elements of a given gene nucleates the assembly of multiprotein complexes where various forms of functional interactions take place (1). One of the most prevalent is the more-than-additive or synergistic response resulting from the recruitment of an activator to multiple copies of a recognition site (compound response element). Despite their importance, the mechanisms that control synergistic effects are poorly understood (2).We have identified a short regulatory motif embedded in a number of sequence-specific regulators that is both necessary and sufficient to limit their transcriptional synergy (3). Disruption of these conserved synergy control (SC) motifs selectively enhances synergistic activation at compound response elements without altering the activity driven from a single site. Structure͞ function analysis of nine SC motifs from the glucocorticoid (GR), mineralocorticoid, and androgen receptors as well as from ETS-1 (3) and C͞EBP␣ (4) revealed a common core sequence (I͞V-K-X-E) and the presence of proline residues within 0-3 aa from either or both ends of the core. Such sequences occur in conserved regions of many factors and, in some cases, these regions have negative regulatory functions (e.g., SP3, SREBP, c-Myb, and C͞EBP). Moreover, a human mutation (P390S) in one of the SC motifs of the androgen receptor is associated with impaired spermatogenesis (5).A clue to the critical role of the Lys residues in SC motifs came from the identification of the consensus motif (⌿-K-x-E͞D) for the posttranslational modification by SUMO. Conjugation of this ubiquitin-like protein follows an analogous pathway to that of ubiquitination and requires dedicated E1 activating (SAE1͞ SAE2) and E2 conjugating ...