Single-Cell Imaging of Intracellular Ca 2ϩ and Phospholipase C Activity Reveals That RGS 2, 3, and 4 Differentially Regulate Signaling via the G␣ q/11 -Linked Muscarinic M 3 Receptor

ABSTRACT Using single cell, real-time imaging, this study compared the impact of members of the B/R4 subfamily of the regulators of G-protein signaling (RGS) (RGS2, -3, and -4) on receptor-mediated inositol 1,4,5-trisphosphate [Ins(1,4,5)P 3 ], diacylglycerol, and Ca 2ϩ signaling. In human embryonic kidney (HEK) 293 cells expressing recombinant G␣ q/11 -coupled muscarinic M 3 receptors, transient coexpression of RGS proteins with fluorescentlytagged biosensors for either Ins(1,4,5)P 3 or diacylglycerol demonstrated that RGS2 and 3 inhibited receptor-mediated events. Although gross indices of signaling were unaffected by RGS4, it slowed the rate of increase in Ins(1,4,5)P 3 levels. At equivalent levels of expression, myc-tagged RGS proteins showed inhibitory activity on the order RGS3 Ն RGS2 Ͼ RGS4. In HEK293 cells, stable expression of myc-tagged RGS2, -3, or -4 at equivalent levels also inhibited phosphoinositide and Ca 2ϩ signaling by endogenously expressed muscarinic M 3 receptors in the order RGS3 Ն RGS2 Ͼ RGS4. In these cells, RGS2 or -3 reduced receptor-mediated inositol phosphate generation in cell populations and reduced both the magnitude and kinetics (rise-time) of single cell Ca 2ϩ signals. Furthermore, at low levels of receptor activation, oscillatory Ca 2ϩ signals were dampened or abolished, whereas at higher levels, RGS2 and -3 promoted the conversion of more stable Ca 2ϩ elevations into oscillatory signals. Despite little or no effect on responses to maximal receptor activation, RGS4 produced effects on the magnitude, kinetics, and oscillatory behavior of Ca 2ϩ signaling at submaximal levels that were consistent with those of RGS2 and -3. The family of regulators of G-protein signaling (RGS) negatively regulate signaling by G-protein-coupled receptors (GPCRs) by binding to activated G␣-subunits and acting as either GTPase-activating proteins (GAPs) or effector antagonists RGS and RGS-like proteins have been classified into subfamilies based on the alignment of the RGS domain amino acid sequence

Desensitization of Gonadotropin-releasing Hormone Action in αT3-1 Cells Due to Uncoupling of Inositol 1,4,5-Trisphosphate Generation and Ca 2+ Mobilization

Gonadotropin-releasing hormone (GnRH) acts via a G-protein coupled receptor on gonadotropes to increase cytosolic Ca2+ and stimulate gonadotropin secretion. Sustained exposure causes desensitization of these effects, but the GnRH receptor has no C-terminal tail and does not undergo rapid (<5 min) desensitization. Nevertheless, pretreatment of alphaT3-1 cells with GnRH reduced the spike Ca2+ response to GnRH and decreased the GnRH effect on inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) by 30-50%. Ca2+-free medium with or without thapsigargin also decreased GnRH-stimulated Ins(1,4,5)P3 generation, implying that attenuation of the Ca2+ response underlies the Ins(1,4,5)P3 reduction rather than vice versa. Intracellular Ca2+ pool depletion cannot explain desensitization of the Ca2+ response because pool depletion and repletion were faster (half-times, <1 min) than the onset of and recovery from desensitization (half-times 10-20 min and 4-6 h). Moreover, 1-h GnRH pre-treatment attenuated the spike Ca2+ response to GnRH but not that to ionomycin, and brief GnRH exposure in Ca2+-free medium reduced the response to ionomycin more effectively in controls than in desensitized cells. GnRH pretreatment also attenuated the Ca2+ response to PACAP38. This novel form of desensitization does not reflect uncoupling of GnRH receptors from their immediate effector system but rather a reduced efficiency of mobilization by Ins(1,4,5)P3 of Ca2+ from an intact intracellular pool

Molecular architecture of Gαo and the structural basis for RGS16-mediated deactivation

Heterotrimeric G proteins relay extracellular cues from heptahelical transmembrane receptors to downstream effector molecules. Composed of an α subunit with intrinsic GTPase activity and a βγ heterodimer, the trimeric complex dissociates upon receptor-mediated nucleotide exchange on the α subunit, enabling each component to engage downstream effector targets for either activation or inhibition as dictated in a particular pathway. To mitigate excessive effector engagement and concomitant signal transmission, the Gα subunit's intrinsic activation timer (the rate of GTP hydrolysis) is regulated spatially and temporally by a class of GTPase accelerating proteins (GAPs) known as the regulator of G protein signaling (RGS) family. The array of G protein-coupled receptors, Gα subunits, RGS proteins and downstream effectors in mammalian systems is vast. Understanding the molecular determinants of specificity is critical for a comprehensive mapping of the G protein system. Here, we present the 2.9 Å crystal structure of the enigmatic, neuronal G protein Gαo in the GTP hydrolytic transition state, complexed with RGS16. Comparison with the 1.89 Å structure of apo-RGS16, also presented here, reveals plasticity upon Gαo binding, the determinants for GAP activity, and the structurally unique features of Gαo that likely distinguish it physiologically from other members of the larger Gαi family, affording insight to receptor, GAP and effector specificity

Education and self-management for people newly diagnosed with type 2 diabetes: a qualitative study of patients' views.

OObjectives: We explored the perceptions, views and experiences of diabetes education in people with type 2 diabetes who were participating in a UK randomized controlled trial of methods of education. The intervention arm of the trial was based on DESMOND, a structured programme of group education sessions aimed at enabling self-management of diabetes, while the standard arm was usual care from general practices. Methods: Individual semi-structured interviews were conducted with 36 adult patients, of whom 19 had attended DESMOND education sessions and 17 had been randomized to receive usual care. Data analysis was based on the constant comparative method. Results: Four principal orientations towards diabetes and its management were identified: `resisters', `identity resisters, consequence accepters', `identity accepters, consequence resisters' and `accepters'. Participants offered varying accounts of the degree of personal responsibility that needed to be assumed in response to the diagnosis. Preferences for different styles of education were also expressed, with many reporting that they enjoyed and benefited from group education, although some reported ambivalence or disappointment with their experiences of education. It was difficult to identify striking thematic differences between accounts of people on different arms of the trial, although there was some very tentative evidence that those who attended DESMOND were more accepting of a changed identity and its implications for their management of diabetes. Discussion: No one single approach to education is likely to suit all people newly diagnosed with diabetes, although structured group education may suit many. This paper identifies varying orientations and preferences of people with diabetes towards forms of both education and self-management, which should be taken into account when planning approaches to education