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.

Regulator of G protein signaling (RGS) proteins negatively regulate receptor-mediated second messenger responses by enhancing the GTPase activity of G␣ subunits. We describe a receptor-specific role for an RGS protein at the level of an individual brain neuron. RGS9-2 and G␤5 mRNA and protein complexes were detected in striatal cholinergic and ␥-aminobutyric acidergic neurons. Dialysis of cholinergic neurons with RGS9 constructs enhanced basal Ca 2؉ channel currents and reduced D2 dopamine receptor modulation of Cav2.2 channels. These constructs did not alter M2 muscarinic receptor modulation of Cav2.2 currents in the same neuron. The noncatalytic DEP-GGL domain of RGS9 antagonized endogenous RGS9-2 activity, enhancing D2 receptor modulation of Ca 2؉ currents. In vitro, RGS9 constructs accelerated GTPase activity, in agreement with electrophysiological measurements, and did so more effectively at Go than Gi. These results implicate RGS9-2 as a specific regulator of dopamine receptor-mediated signaling in the striatum and identify a role for GAP activity modulation by the DEP-GGL domain.calcium ͉ GTPase activating protein ͉ receptor-specific ͉ basal ganglia ͉ indirect pathway R egulators of G protein signaling (RGS) are a diverse family of proteins identified by the presence of a 120-aa domain termed the RGS box. In cell lines or in purified in vitro assays, most RGS proteins enhance the GTPase activity of heterotrimeric G protein ␣ subunits and thereby accelerate the deactivation of receptor-initiated second messenger responses. Many RGS proteins also contain one or more putative protein-protein interaction domains. These noncatalytic domains have been suggested to regulate catalytic activity, signal transduction pathway specificity, and͞or subcellular targeting of RGS proteins.One subfamily of RGS proteins (RGS6, -7, -9, and -11) all share homologous DEP (Dishevelled, Egl-10, Pleckstrin), GGL (G protein Gamma subunit Like), and RGS domains. The DEP domain of the retinal isoform of RGS9 (RGS9-1) has been shown to confer localization to a retinal membrane protein termed R9AP, and this localization has been shown to be necessary for proper RGS9-1 function in the retina (1). Several investigators have demonstrated that the GGL domain interacts with the G␤ 5 subunit (2-6). In vitro, G␤ 5 binding to RGS6, -7, or -11 increases the GAP specificity of these RGS proteins for G␣ (2, 3). In addition, G␤ 5 binding to either RGS7 or RGS9 enhances RGS-mediated acceleration of G protein gated inwardly rectifying K ϩ (Kir3) channel activation and deactivation kinetics in an oocyte expression system. This enhancement may result from G␤ 5 -mediated increased stability of the RGS protein or enhanced GAP activity (7).Despite the functional similarities among RGS6, -7, -9, and -11 in heterologous expression systems or when analyzed in vitro, each of these RGS proteins is likely to play a unique role in the central nervous system because they are differentially localized within the brain (8, 9). In contrast to the more ubiquitous loc...

We previously reported that residues 299 -318 in G␣ i1 participate in the selective interaction between G␣ i1 and the 5-hydroxytryptamine 1B (5-HT 1B ) receptor (Bae, H., Anderson, K., Flood, L. A., Skiba, N. P., Hamm, H. E., and Graber, S. G. (1997) J. Biol. Chem. 272, 32071-32077). The present study more precisely defines which residues within this domain are critical for 5-HT 1B receptormediated G protein activation. A series of G␣ i1 /G␣ t chimeras and point mutations were reconstituted with G␤␥ and Sf9 cell membranes containing the 5-HT 1B receptor. Functional coupling to 5-HT 1B receptors was assessed by 1) [35 S]GTP␥S binding and 2) agonist affinity shift assays. Replacement of the ␣4 helix of G␣ i1 (residues 299 -308) with the corresponding sequence from G␣ t produced a chimera (Chi22) that only weakly coupled to the 5-HT 1B receptor. In contrast, substitution of residues within the ␣4-␤6 loop region of G␣ i1 (residues 309 -318) with the corresponding sequence in G␣ t either permitted full 5-HT 1B receptor coupling to the chimera (Chi24) or only minimally reduced coupling to the chimeric protein (Chi25). Two mutations within the ␣4 helix of G␣ i1 (Q304K and E308L) reduced agonist-stimulated [ 35 S]GTP␥S binding, and the effects of these mutations were additive. The opposite substitutions within Chi22 (K300Q and L304E) restored 5-HT 1B receptor coupling, and again the effects of the two mutations were additive. Mutations of other residues within the ␣4 helix of G␣ i1 had minimal to no effect on 5-HT 1B coupling behavior. These data provide evidence that ␣4 helix residues in G␣ i participate in directing specific receptor interactions and suggest that Gln 304 and Glu 308 of G␣ i1 act in concert to mediate the ability of the 5-HT 1B receptor to couple specifically to inhibitory G proteins.The interaction of heptahelical receptors with their cognate heterotrimeric guanine nucleotide-binding proteins (G proteins) represents an initial step in the transmission of extracellular signals across the plasma membrane (2-4). The receptor-G protein interaction modulates specific second messenger systems that result in a unique physiologic response to the extracellular signal. The particular downstream effect of G protein activation is not the result of an explicit interaction between each heptahelical receptor and a unique heterotrimeric G protein. On the contrary, G protein-coupled receptors have repeatedly been demonstrated to couple to several related members within the same family of G protein ␣ subunits, albeit with differing levels of efficiency (5-11). Clawges et al. (12) demonstrated that the serotonin (5-HT) 1 1B receptor couples to heterotrimers containing either G␣ i1 , G␣ i2 , G␣ i3 , or G␣ o . Nevertheless, this receptor does not couple to heterotrimers containing another member of this same family, the G␣ t subunit (12). Therefore, the 5-HT 1B receptor represents one receptor system that can be exploited to investigate the precise molecular determinants governing selective receptor-G protein interactio...

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...

This study assesses the efficacy of cognitive behaviour therapy (CBT) in treating premenstrual dysphoric changes. The CBT condition (n = 24) aimed to modify dysfunctional thinking as a means of impacting on negative premenstrual symptoms and changes. The components of CBT were cognitive restructuring and assertion training. A comparison condition called 'information-focused therapy' (IFT) (n = 9) aimed to present information only and did not address belief restructuring. The components were relaxation training, nutritional and vitamin guidelines, dietary and lifestyle recommendations, aspects of child management training and assertion training. Results indicated that the amelioration of anxiety, depression, negative thoughts and physical changes can be effectively addressed by either CBT or IFT. The extent to which a woman's belief system is critical in the experience of premenstrual distress requires further empirical investigation.

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We have reported previously prenatal cocaine-induced functional deficits in serotonergic terminals, and gender-specific supersensitivity of postsynaptic 5-HT(1A) receptor-mediated hormone responses in offspring. This study investigates the effects of prenatal exposure to cocaine on postsynaptic 5-HT(2A) receptor-mediated responses in prepubescent male and female offspring. Pregnant rats were administered saline or (-)cocaine (15 mg/kg, s.c., b.i.d) from gestational day 13 through 20. Changes in 5-HT(2A) receptor function in offspring were assessed by differences in the ability of DOI [4-iodo, 2,5-dimethoxyphenyl-isopropylamine; 2. 0 mg/kg, s.c.] to elevate plasma levels of the hormones ACTH, corticosterone and renin. Basal hormone levels in male and female progeny were unaffected by prenatal cocaine exposure. However, prenatal exposure to cocaine significantly potentiated the magnitude of the ACTH response to DOI in both male (+19%) and female (+43%) progeny. Similarly, the DOI-induced elevation of plasma renin was markedly potentiated in male (+51%) and female (+83%) cocaine-exposed offspring. Although DOI significantly elevated corticosterone levels in both male and female offspring, the magnitude of corticosterone responses was not altered by prenatal exposure to cocaine. Densities of agonist ((125)I-DOI)-labeled receptors in hypothalamus and cortex were unaltered by prenatal exposure to cocaine. These data indicate prenatal cocaine-induced supersensitivity of postsynaptic 5-HT(2A) receptor function in male and female offspring without changes in receptor density. Synapse: 35:163-172, 2000.