Accessory proteins involved in signal processing through heterotrimeric G proteins are generally defined as proteins distinct from G protein-coupled receptor (GPCR), G protein, or classical effectors that regulate the strength/efficiency/specificity of signal transfer upon receptor activation or position these entities in the right microenvironment, contributing to the formation of a functional signal transduction complex. A flurry of recent studies have implicated an additional class of accessory proteins for this system that provide signal input to heterotrimeric G proteins in the absence of a cell surface receptor, serve as alternative binding partners for G protein subunits, provide unexpected modes of G protein regulation, and have introduced additional functional roles for G proteins. This group of accessory proteins includes the recently discovered Activators of G protein Signaling (AGS) proteins identified in a functional screen for receptor-independent activators of G protein signaling as well as several proteins identified in protein interaction screens and genetic screens in model organisms. These accessory proteins may influence GDP dissociation and nucleotide exchange at the G(alpha) subunit, alter subunit interactions within heterotrimeric G(alphabetagamma) independent of nucleotide exchange, or form complexes with G(alpha) or G(betagamma) independent of the typical G(alphabetagamma) heterotrimer. AGS and related accessory proteins reveal unexpected diversity in G protein subunits as signal transducers within the cell.

GnRH regulates the reproductive system by stimulating synthesis and release of gonadotropins. GnRH acts through a receptor coupled to multiple intracellular events including a rapid phosphoinositide turnover. Although the cAMP pathway is essential for gonadotrope function, the ability of GnRH to induce cAMP, as well as the coupling mechanisms involved, remain controversial. In this study, we established that GnRH increases intracellular cAMP levels in a concentration-dependent manner in LbetaT2 gonadotrope cells (maximal increase, 2.5-fold; EC(50), 0.30 nm), and this was further evidenced by GnRH activation of a cAMP-sensitive reporter gene. The GnRH effect was Ca(2+) independent, mimicked by the phorbol ester phorbol 12-myristate 13-acetate, and blocked by the protein kinase C (PKC) inhibitor bisindolylmaleimide, indicating that the GnRH effect was mediated by PKC. Pharmacological inhibition of conventional PKC isoforms with Gö6976 did not prevent GnRH-induced cAMP production, whereas down-regulation of novel PKCdelta, -epsilon, and -theta by a long-term treatment with GnRH markedly reduced it. Expression of dominant-negative (DN) mutants of PKCdelta or -epsilon but not PKCtheta impaired GnRH activation of a cAMP-sensitive promoter, demonstrating that PKCdelta and -epsilon are the two endogenous isoforms mediating GnRH activation of the adenylyl cyclase (AC) pathway in LbetaT2 cells. Accordingly, we identified by RT-PCR and immunocytochemical analysis, two PKC-sensitive AC isoforms, i.e. AC5 and AC7 as potential targets for GnRH. Lastly, we showed that only sustained stimulation of GnRH receptor significantly increased cAMP, suggesting that in vivo, the cAMP signaling pathway may be selectively recruited under intense GnRH release such as the preovulatory GnRH surge.

The activity of pituitary gonadotrope cells, crucial for reproductive function, is regulated by numerous factors including signals related to nutritional status. In this work, we demonstrated, for the first time, that in vivo central exposure of rats to lipids intracarotid infusion of a heparinized triglyceride emulsion selectively increases the expression of pituitary LH subunit genes without any alteration of pituitary GnRH receptor and hypothalamic GnRH or Kiss-1 transcript levels. Furthermore, we showed that unsaturated fatty acids (UFA), oleate and linoleate, increase LH release in a dose-dependent manner as well as LHβ mRNA levels in both immortalized LβT2 gonadotrope cell line and rat primary cell cultures. In contrast, the saturated palmitate was ineffective. ACTH or TSH secretion was unaffected by UFA treatment. We demonstrated in LβT2 cells that linoleate effect is mediated neither by activation of membrane fatty acid (FA) receptors GPR40 or GPR120 although we characterized these receptors in LβT2 cells, nor through nuclear peroxisome proliferator-activated receptors. Furthermore, linoleate β-oxidation is not required for its action on LH secretion. In contrast, pharmacological inhibition of protein kinase C (PKC) or ERK pathways significantly prevented linoleate-stimulated LH release. Accordingly, linoleate was shown to activate novel PKC isoforms, PKCε and -θ, as well as ERK1/2 in LβT2 cells. Lastly, unsaturated, but not saturated, FA inhibited GnRH-induced LH secretion in LβT2 cells as well as in pituitary cell cultures. Altogether, these results suggest that the pituitary is a relevant site of FA action and that UFA may influence reproduction by directly interfering with basal and GnRH-dependent gonadotrope activity.

AGS3, a receptor-independent activator of G-protein signaling, is involved in unexpected functional diversity for G-protein signaling systems. AGS3 has seven tetratricopeptide (TPR) motifs upstream of four G-protein regulatory (GPR) motifs that serve as docking sites for Gi␣-GDP. The positioning of AGS3 within the cell and the intramolecular dynamics between different domains of the proteins are likely key determinants of their ability to influence G-protein signaling. We report that AGS3 enters into the aggresome pathway and that distribution of the protein is regulated by the AGS3 binding partners Gi␣ and mammalian Inscuteable (mInsc). Gi␣ rescues AGS3 from the aggresome, whereas mInsc augments the aggresome-like distribution of AGS3. The distribution of AGS3 to the aggresome is dependent upon the TPR domain, and it is accelerated by disruption of the TPR organizational structure or introduction of a nonsynonymous single-nucleotide polymorphism. These data present AGS3, G-proteins, and mInsc as candidate proteins involved in regulating cellular stress associated with protein-processing pathologies.The discovery of AGS3 (GPSM1) and related accessory proteins revealed unexpected functional diversity for G-protein signaling systems (8,36). AGS3 is involved in a number of different cellular activities, including asymmetric cell division during neuronal development (30), neuronal plasticity and addiction (9, 10, 12, 38, 39), autophagy (27), membrane protein trafficking (17), cardiovascular function (7), and metabolism (7). AGS3 is a multidomain protein consisting of seven tetratricopeptide repeats (TPR) in the amino-terminal portion of the protein and four G-protein regulatory (GPR) motifs in the carboxyl region of the protein. Each of the GPR motifs binds and stabilizes the GDP-bound conformation of G␣ (Gi␣, Gt␣, and Gi/o␣), essentially behaving as a guanine nucleotide dissociation inhibitor. As such, AGS3 may be complexed with up to four G␣ and function as an alternative binding partner for G␣ independently of the classical heterotrimeric G␣␤␥. Despite the clearly demonstrated function of AGS3 and the related protein LGN (GPSM2 or AGS5) in various model organisms and a fairly solid, basic biochemical understanding of the interaction of a GPR motif with G␣, the signals that operate "upstream" and/or "downstream" of AGS3 or an AGS3-Gi/o␣ complex are not well defined.AGS3 and other GPR proteins may regulate G-protein signaling directly by influencing the interaction of G␣ with G␤␥ or another G␣ binding partner. In addition, a portion of G␣ in the cell is complexed with GPR proteins to various degrees, and this interaction is regulated. Ric-8A interacts with an AGS3-Gi␣ complex in a manner somewhat analogous to the interaction of a G-protein-coupled receptor with heterotrimeric G␣␤␥, promoting nucleotide exchange and the apparent dissociation of AGS3 and Gi␣-GDP (37). The specific impact of AGS3 and other GPR proteins on signaling events is likely dependent upon where the individual protein is positioned within the c...