Strategy for the Thermostabilization of an Agonist-Bound GPCR Coupled to a G Protein.

Structure determination of G protein-coupled receptors (GPCRs) in the inactive state bound to high-affinity antagonists has been very successful through the implementation of a number of protein engineering and crystallization strategies. However, the structure determination of GPCRs in their fully active state coupled to a G protein is still very challenging. Recently, mini-G proteins were developed, which recapitulate the coupling of a full heterotrimeric G protein to a GPCR despite being less than one-third of the size. This allowed the structure determination of the agonist-bound adenosine A2A receptor (A2AR) coupled to mini-Gs. Although this is extremely encouraging, A2AR is very stable compared with many other GPCRs, particularly when an agonist is bound. In contrast, the agonist-bound conformation of the human corticotropin-releasing factor receptor is considerably less stable, impeding the formation of good quality crystals for structure determination. We have therefore developed a novel strategy for the thermostabilization of a GPCR-mini-G protein complex. In this chapter, we will describe the theoretical and practical principles of the thermostability assay for stabilizing this complex, discuss its strengths and weaknesses, and show some typical results from the thermostabilization process.

Determination of GPCR Phosphorylation Status: Establishing a Phosphorylation Barcode.

G protein-coupled receptors (GPCRs) are rapidly phosphorylated following agonist occupation in a process that mediates receptor uncoupling from its cognate G protein, a process referred to as desensitization. In addition, this process provides a mechanism by which receptors can engage with arrestin adaptor molecules and couple to downstream signaling pathways. The importance of this regulatory process has been highlighted recently by the understanding that ligands can direct receptor signaling along one pathway in preference to another, the phenomenon of signaling bias that is partly mediated by the phosphorylation status or phosphorylation barcode of the receptor. Methods to determine the phosphorylation status of a GPCR in vitro and in vivo are necessary to understand not only the physiological mechanisms involved in GPCR signaling, but also to fully examine the signaling properties of GPCR ligands. This unit describes detailed methods for determining the overall phosphorylation pattern on a receptor (the phosphorylation barcode), as well as mass spectrometry approaches that can define the precise sites that become phosphorylated. These techniques, coupled with the generation and characterization of receptor phosphorylation-specific antibodies, provide a full palate of techniques necessary to determine the phosphorylation status of any given GPCR subtype.

Neural tube defects and impaired neural progenitor cell proliferation in Gbeta1-deficient mice.

Heterotrimeric G proteins are well known for their roles in signal transduction downstream of G protein-coupled receptors (GPCRs), and both Galpha subunits and tightly associated Gbetagamma subunits regulate downstream effector molecules. Compared to Galpha subunits, the physiological roles of individual Gbeta and Ggamma subunits are poorly understood. In this study, we generated mice deficient in the Gbeta1 gene and found that Gbeta1 is required for neural tube closure, neural progenitor cell proliferation, and neonatal development. About 40% Gbeta1(-/-) embryos developed neural tube defects (NTDs) and abnormal actin organization was observed in the basal side of neuroepithelium. In addition, Gbeta1(-/-) embryos without NTDs showed microencephaly and died within 2 days after birth. GPCR agonist-induced ERK phosphorylation, cell proliferation, and cell spreading, which were all found to be regulated by Galphai and Gbetagamma signaling, were abnormal in Gbeta1(-/-) neural progenitor cells. These data indicate that Gbeta1 is required for normal embryonic neurogenesis.

[The receptor of serpentine type and the heterotrimeric G protein as targets of action of the polylysine dendrimers].

The molecular mechanisms of action of the polycationic peptides--polylysine homo- and heterodendrimers on functional activity of biogenic amines- and peptide hormones-sensitive adenylyl; cyclase signaling system (AC system) in the myocardium and the brain of rats were studied. These peptides are expected to be used as highly effective polymer carries for biologically active substances. The polylysine homodendrimers of the third [(NH2)16(Lys)8(Lys)4(Lys)2Lys-Ala-NH2] (I), fourth [(NH2)32(Lys)16(Lys)8(Lys)4(Lys)2Lys-Ala-NH2 (II) and fifth [(NH2)64(Lys)32(Lys)16(Lys)8(Lys)4(Lys)2Lys-Ala-NH2] (III) generations and the polylysine homodendrimers of fifth generation--[(NH2)64(Lys-Glu)32(Lys-Glu)16(Lys-Glu)8(Lys-Glu)4(Lys-Glu)2Lys-Ala-Ala-Lys (ClAc)-Ala-NH2] (IV), [(NH2)64(Lys-Ala)32(Lys-Ala)16(Lys-Ala)8(Lys-Ala)4(Lys-Ala)2Lys-Ala-Lys(ClAc)-Ala-Ala-NH2] (V) and [(NH2)64(Lys-Gly-Gly)32(Lys-Gly-Gly)16(Lys-Gly-Gly)8(Lys-Gly-Gly)4(Lys-Gly-Gly)2 Lys-Gly-Gly-Lys(ClAc)-Ala-Ala-NH2] (VI) showed receptor-independent mechanism of heterotrimeric G-proteins activity, preferably of inhibitory type, interacting with C-terminal regions of their alpha-subunits. The homodendrimers II and III and heterodendrimer V are more effective G-protein activators. The polylysine dendrimers disturbed the functional coupling of the receptors of biogenic amines and peptides hormones with Gi-proteins and, in a lesser extent, Gs-proteins. This is illustrated by the decrease in regulatory effects of the hormones on AX activity and G-protein GTP binding and by the decrease in receptor affinity to agonists in the presence of the polylysine dendrimers, as result of receptor--G-proteins complex dissociation. It was shown also that the molecular mechanisms and the selectivity of the action on the G-proteins of the polylysine dendrimers were similar to those of mastoparan and melittin, natural toxins of insect venom.

Cardiac-specific overexpression of diacylglycerol kinase zeta prevents Gq protein-coupled receptor agonist-induced cardiac hypertrophy in transgenic mice.

BACKGROUND: Diacylglycerol is a lipid second messenger that accumulates in cardiomyocytes when stimulated by Gqalpha protein-coupled receptor (GPCR) agonists such as angiotensin II, phenylephrine, and others. Diacylglycerol functions as a potent activator of protein kinase C (PKC) and is catalyzed by diacylglycerol kinase (DGK) to form phosphatidic acid and inactivated. However, the functional roles of DGK have not been previously examined in the heart. We hypothesized that DGK might prevent GPCR agonist-induced activation of diacylglycerol downstream signaling cascades and subsequent cardiac hypertrophy. METHODS AND RESULTS: To test this hypothesis, we generated transgenic (DGKzeta-TG) mice with cardiac-specific overexpression of DGKzeta. There were no differences in heart size and heart weight between DGKzeta-TG and wild-type littermate mice. The left ventricular function was normal in DGKzeta-TG mice. Continuous administration of subpressor doses of angiotensin II and phenylephrine caused PKC translocation, gene induction of atrial natriuretic factor, and subsequent cardiac hypertrophy in WT mice. However, in DGKzeta-TG mice, neither translocation of PKC nor upregulation of atrial natriuretic factor gene expression was observed after angiotensin II and phenylephrine infusion. Furthermore, in DGKzeta-TG mice, angiotensin II and phenylephrine failed to increase cross-sectional cardiomyocyte areas and heart to body weight ratios. Phenylephrine-induced increases in myocardial diacylglycerol levels were completely blocked in DGKzeta-TG mouse hearts, suggesting that DGKzeta regulated PKC activity by controlling cellular diacylglycerol levels. CONCLUSIONS: These results demonstrated the first evidence that DGKzeta negatively regulated the hypertrophic signaling cascade and resultant cardiac hypertrophy in response to GPCR agonists without detectable adverse effects in in vivo hearts.

Coupling of the human A1 adenosine receptor to different heterotrimeric G proteins: evidence for agonist-specific G protein activation.

The present study investigates the effect of varying ligand structure on the ability of agonists to activate guanine nucleotide-binding proteins of the Gi, Gs and Gq families via the A(1) adenosine receptor. In CHO cells expressing this receptor, inhibition or potentiation of forskolin-stimulated cAMP accumulation was used as an end point to measure the activation of Gi and, in Pertussis toxin (PTX)-treated cells, Gs, respectively. Stimulation of inositol phosphate accumulation in PTX-treated cells was used as an index of Gq activation. CPA (N(6)-cyclopentyladenosine), NECA (5'-N-ethyl-carboxyamidoadenosine) and eight analogues of these ligands presented a range of guanine nucleotide-binding protein (G-protein)-activating profiles. Some ligands could only activate Gi (e.g. 2'deoxyCPA), some primarily Gi and Gs (and only weakly Gq) (e.g. 3'deoxyCPA), highlighting the importance of the ribose hydroxyls in agonist activation of multiple G proteins. CHA (N(6)-cyclohexyladenosine) activated Gi, Gs and Gq, but was more efficacious than CPA in activating Gs. The NECA analogues 5'-N-cyclopropyl-carboxamidoadenosine, 5'-N-cyclobutyl-carboxamidoadenosine and 5'-N-cyclopentyl-carboxamidoadenosine (CPeCA) also activated all three G proteins, although their ability to activate Gs and Gq (relative to CPA) was reduced with increasing substituent size, such that CPeCA produced only a small stimulation (at 100 microM) at Gq, but was a full agonist, relative to CPA, at Gi and Gs. This study suggests that the A(1) adenosine receptor can adopt agonist-specific conformations, arising from small changes in ligand structure, which lead to the differential activation of Gi, Gs and Gq.

Analyses of RGS protein control of agonist-evoked Ca2+ signaling.

Analysis of the function of regulator of G-protein signaling (RGS) protein function and their selectivity of action in vivo is complicated by the expression of multiple RGS proteins in a single cell and requires precise control of cytosolic RGS protein concentration. This article describes two experimental systems using pancreatic acinar cells suitable for such analyses. The first is pancreatic acini permeabilized with streptolysin O, which retains agonist responsiveness while allowing RGS proteins and molecules with molecular masses of up to 25-30 kDa access to the cytosol. The second is a whole cell recording of the Ca(2+)-activated Cl- current of single pancreatic acinar cells as a reporter of [Ca2+]i. This system can be used to introduce to the cytosol any protein of interest, including recombinant RGS proteins and RGS protein-scavenging antibodies. The use of these systems to study the specificity of RGS proteins action, the function of their domains, and the role of RGS proteins in controlling Ca2+ oscillations is discussed.

Functional coupling of the human dopamine D2 receptor with G alpha i1, G alpha i2, G alpha i3 and G alpha o G proteins: evidence for agonist regulation of G protein selectivity.

(1) The human dopamine D(2long) (D(2L)) receptor was expressed with four different G proteins in Sf9 cells using the baculovirus expression system. When co-expressed with G(i)/G(o) G proteins (G(i1)alpha, G(i2)alpha, G(i3)alpha, or G(o)alpha, plus Gbeta(1) and Ggamma(2)), the receptor displayed a high-affinity binding site for the agonists (dopamine and NPA), which was sensitive to GTP (100 micro M), demonstrating interaction between the receptor and the different G proteins. (2) The receptor to G protein ratio (R : G ratio) was evaluated using [(3)H]-spiperone saturation binding (R) and [(35)S]-GTPgammaS saturation binding (G). R : G ratios of 1 : 12, 1 : 3, 1 : 14 and 1 : 5 were found for G(i1), G(i2), G(i3), and G(o) preparations, respectively. However, when R : G ratios of 1 : 2 and 1 : 12 were compared for G(i2) and G(o), no difference was found for the stimulation of [(35)S]-GTPgammaS binding. (3) Several agonists were tested for their ability to stimulate [(35)S]-GTPgammaS binding to membranes co-expressing the receptor and various G proteins. All the compounds tested showed agonist activity in preparations expressing G(i3) and G(o). However, for G(i2) and G(i1) preparations, compounds such as S-(-)-3-PPP and p-tyramine were unable to stimulate [(35)S]-GTPgammaS binding. (4) Most of the compounds showed higher relative efficacies (compared to dopamine) and higher potencies in the preparation expressing G(o). Comparison of the effects of different agonists in the different preparations showed that each agonist differentially activates the four G proteins. (5) We conclude that the degree of selectivity of G protein activation by the D(2L) receptor can depend on the conformation of the receptor stabilised by an agonist.

Nod factors activate both heterotrimeric and monomeric G-proteins in Vigna unguiculata (L.) Walp.

Nod factors are lipo-chito-oligosaccharides secreted by rhizobia that initiate many responses in the root hairs of the legume hosts, culminating in deformed hairs. The heterotrimeric G-protein agonists mastoparan, Mas7, melittin, compound 48/80 and cholera toxin provoke root hair deformation, whereas the heterotrimeric G-protein antagonist pertussis toxin inhibits mastoparan and Nod factor NodNGR[S]- (from Rhizobiumsp. NGR234) induced root hair deformation. Another heterotrimeric G-protein antagonist, isotetrandrine, only inhibited root hair deformation provoked by mastoparan and melittin. These results support the notion that G-proteins are implicated in Nod factor signalling. To study the role of G-proteins at a biochemical level, we examined the GTP-binding profiles of root microsomal membrane fractions isolated from the nodulation competent zone of Vigna unguiculata(L.) Walp. GTP competitively bound to the microsomal membrane fractions labelled with [(35)S]GTPgammaS, yielding a two-site displacement curve with displacement constants ( K(i)) of 0.58 micro M and 0.16 mM. Competition with either ATP or GDP revealed a one-site displacement curve with K(i) of 4.4 and 29 micro M, respectively, whereas ADP and UTP were ineffective competitors. The GTP-binding profiles of microsomal membrane fractions isolated from roots pretreated with either NodNGR[S] or the four-sugar, N- N'- N"- N'"-tetracetylchitotetraose (TACT) backbone of Nod factors were significantly altered compared with control microsomal fractions. To identify candidate proteins, membrane proteins were separated by SDS-PAGE and electrotransferred to nitrocellulose. GTP overlay experiments revealed that membrane fractions isolated from roots pretreated with NodNGR[S] or TACT contained two proteins (28 kDa and 25 kDa) with a higher affinity for GTPgammaS than control membrane fractions. Western analysis demonstrated that membranes from the pretreated roots contained more of another protein (~55 kDa) recognised by Galpha(common) antisera. These results provide pharmacological and biochemical evidence supporting the contention that G-proteins are involved in Nod factor signalling and, importantly, implicate monomeric G-proteins in this process.

Gq-coupled receptor agonists mediate cardiac hypertrophy via the vasculature.

The Gq-coupled receptor-signaling pathway has been implicated in the cardiac hypertrophic response to stress, but little is actually known about the contributions of Gq signaling in either the heart or the vasculature. Therefore, we developed a line of transgenic mice that express a peptide inhibitor of Gq (GqI) in vascular smooth muscle to determine if vascular Gq signaling was important in the cardiac hypertrophic response. After chronic administration of the Gq agonists phenylephrine, serotonin, and angiotensin II, we observed an attenuation of mean arterial blood pressure and an inhibition of cardiac hypertrophy in the transgenic mice with vascular-specific GqI expression. In contrast, cardiac GqI peptide expression did not attenuate the hypertension or the cardiac hypertrophy. Importantly, all mice were capable of cardiac hypertrophy, because direct beta-adrenergic receptor stimulation induced a similar level of hypertrophy in both lines of transgenic mice. This clearly suggests that after chronic Gq-coupled receptor agonist administration, it is the hypertensive state induced by vascular Gq activation that mediates remodeling of the heart, rather than direct stimulation of cardiac Gq-coupled receptors. Thus, the contribution of vascular Gq-coupled signaling to the development of cardiac hypertrophy is significant and suggests that expression of the GqI peptide is a novel therapeutic strategy to lower Gq-mediated hypertension and cardiac hypertrophy.

Dual actions of the Galpha(q) agonist Pasteurella multocida toxin to promote cardiomyocyte hypertrophy and enhance apoptosis susceptibility.

Previous attempts to delineate the consequences of Galpha (q) activation in cardiomyocytes relied largely on molecular strategies in cultures or transgenic mice. Modest levels of wild-type Galpha(q) overexpression induce stable cardiac hypertrophy, whereas intense Galpha(q) stimulation induces cardiomyocyte apoptosis. The precise mechanism(s) whereby traditional targets of Galpha (q) subunits that induce hypertrophy also trigger cardiomyocyte apoptosis is not obvious and is explored with recombinant Pasteurella multocida toxin (rPMT, a Galpha(q) agonist). Cells cultured with rPMT display cardiomyocyte enlargement, sarcomeric organization, and increased atrial natriuretic factor expression in association with activation of phospholipase C, novel protein kinase C (PKC) isoforms, extracellular signal-regulated protein kinase (ERK), and (to a lesser extent) JNK/p38-MAPK. rPMT stimulates the ERK cascade via epidermal growth factor (EGF) receptor transactivation in cardiac fibroblasts, but EGF receptor transactivation plays no role in ERK activation in cardiomyocytes. Surprisingly, rPMT (or novel PKC isoform activation by PMA) decreases basal Akt phosphorylation; rPMT prevents Akt phosphorylation by EGF or IGF-1 and functionally augments cardiomyocyte apoptosis in response to H2O2. These results identify a Galpha(q)-PKC pathway that represses basal Akt phosphorylation and impairs Akt stimulation by survival factors. Because inhibition of Akt enhances cardiomyocyte susceptibility to apoptosis, this pathway is predicted to contribute to the transition from hypertrophy to cardiac decompensation and could be targeted for therapy in heart failure.

Effects of age, postmortem delay and storage time on receptor-mediated activation of G-proteins in human brain.

The influence of age, postmortem delay and freezing storage period on receptor-mediated G-protein activity was quantified in cortical membranes from 34 healthy subjects. Concentration-response curves of the [(35)S]GTPgammaS binding stimulation by agonists for alpha(2)-adrenoceptors (UK14304), mu-opioid (DAMGO), 5-HT(1A) (8-OH-DPAT), GABA(B) (baclofen) and muscarinic (carbachol) receptors were analyzed. Immunoreactivities of G(alpha)-protein subunits were also determined. Basal binding and UK14304, 8-OH-DPAT, and baclofen potency to stimulate [(35)S]GTPgammaS binding decreased with aging (1-88 years) without changes of efficacy. DAMGO-mediated stimulation increased both in potency and efficacy with aging. A negative correlation between age and immunoreactivity was observed for G(alphai1/2)-, but not for G(alphai3)-, G(alphao)-,and G(alphas)-proteins. Neither [(35)S]GTPgammaS binding nor G(alpha)-proteins changed with the postmortem delay (8-92 h). Basal [(35)S]GTPgammaS binding decreased with the sample storage period (1-85 months). A careful match between cases and controls should be taken into account when designing signal transduction studies in human disorders, specially for variables such as age and storage period.

MEKK1 is essential for cardiac hypertrophy and dysfunction induced by Gq.

Signaling via mitogen-activated protein kinases is implicated in heart failure induced by agonists for G protein-coupled receptors that act via the G protein Galphaq. However, this assertion relies heavily on pharmacological inhibitors and dominant-interfering proteins and not on gene deletion. Here, we show that endogenous cardiac MAPK/ERK kinase kinase-1 (MEKK1)/(MAP3K1), a mitogen-activated protein kinase kinase kinase, is activated by heart-restricted overexpression of Galphaq in mice. In cardiac myocytes derived from embryonic stem cells in culture, homozygous disruption of MEKK1 selectively impaired c-Jun N-terminal kinase activity in the absence or presence of phenlyephrine, a Galphaq-dependent agonist. Other terminal mitogen-activated protein kinases were unaffected. In mice, the absence of MEKK1 abolished the increase in cardiac mass, myocyte size, hypertrophy-associated atrial natriuretic factor induction, and c-Jun N-terminal kinase activation by Galphaq, and improved ventricular mechanical function. Thus, MEKK1 mediates cardiac hypertrophy induced by Galphaq in vivo and is a logical target for drug development in heart disease involving this pathway.

Receptor-G protein gamma specificity: gamma11 shows unique potency for A(1) adenosine and 5-HT(1A) receptors.

G protein coupled receptors activate signal transducing guanine nucleotide-binding proteins (G proteins), which consist of an alpha subunit and a betagamma dimer. Whole cell studies have reported that receptors signal through specific betagamma subtypes. Membrane reconstitution studies with the adenosine A(1) and alpha(2A) adrenergic receptors have reached a similar conclusion. We aimed to test the generality of this finding by comparing the gamma subtype specificity for four G(i)-coupled receptors: alpha(2A) adrenergic; A1 adenosine (A(1)-R); 5-hydroxytryptamine(1A) (5-HT(1A)-R); mu opioid. Membranes were reconstituted with Galpha(i)(1) and five gamma subtypes (dimerized to beta1). Using a sensitive alpha-betagamma binding assay, we show that all recombinant betagamma (except beta1gamma1) had comparable affinity for alpha(i)(1). Using high affinity agonist binding as a measure of receptor-G protein coupling, betagamma-containing gamma11 was the most potent for A(1)-R and 5-HT(1A)-R (p < 0.05, one way ANOVA) while gamma7 was most potent for the other two receptors. gamma11 was 3-8-fold more potent for the A(1)-R than were the other gamma subtypes. Also, gamma11 was 2-8-fold more potent for A(1)-R than at the other receptors, suggesting a unique coupling specificity of the A(1)-R for gamma11. In contrast, the discrimination by receptors for the other betagamma subtypes (beta1 and gamma1, gamma2, gamma7, and gamma10) was limited (2-3-fold). Thus the exquisite betagamma specificity of individual receptors reported in whole cell studies may depend on in vivo mechanisms beyond direct receptor recognition of betagamma subtypes.

RGS proteins provide biochemical control of agonist-evoked [Ca2+]i oscillations.

Agonist-evoked [Ca2+]i oscillations have been considered a biophysical phenomenon reflecting the regulation of the IP3 receptor by [Ca2+]i. Here we show that [Ca2+]i oscillations are a biochemical phenomenon emanating from regulation of Ca2+ signaling by the regulators of G protein signaling (RGS) proteins. [Ca2+]i oscillations evoked by G protein-coupled receptors require the action of RGS proteins. Inhibition of endogenous RGS protein action disrupted agonist-evoked [Ca2+]i oscillations by a stepwise conversion to a sustained response. Based on these findings and the effect of mutant RGS proteins and anti-RGS protein antibodies on Ca2+ signaling, we propose that RGS proteins within the G protein-coupled receptor complexes provide a biochemical control of [Ca2+]i oscillations.

Modulation of dopamine release in the guinea-pig retina by G(i)- but not by G(s)- or G(q)-protein-coupled receptors.

The modulation of dopamine release from the guinea-pig retina was studied using maximally effective concentrations of 10 agonists acting on G(i)-, G(s)- or G(q)-protein-coupled receptors (PCRs). Retinal discs were preincubated with [(3)H]noradrenaline and superfused; tritium overflow was evoked electrically. The following compounds acting on G(i)-PCRs reduced the tritium overflow, which represents quasi-physiological dopamine release under the experimental conditions of our study: the dopamine and alpha(2)-adrenoceptor agonist B-HT 920 by 95%, the muscarinic agonist oxotremorine by 96%, melatonin by 94%, the cannabinoid agonist WIN 55,212-2 by 71% and histamine by 66%. Tritium overflow was not affected by serotonin or by agonists acting on G(s)-PCRs (ACTH1-24 and the beta-adrenoceptor agonist procaterol) and G(q)-PCRs (angiotensin II and bradykinin). The effects of B-HT 920, oxotremorine and melatonin were studied in more detail using appropriate antagonists. The inhibitory effect of a submaximally active concentration of B-HT 920 was counteracted by the dopamine D(2/3) antagonist haloperidol but not affected by the alpha(2)-adrenoceptor antagonist phentolamine. The muscarinic antagonist atropine shifted to the right the concentration-response curve of oxotremorine (pA(2) 8.7) and the melatonin MT(2) antagonist 4-P-PDOT produced a rightward shift of the concentration-response curve of melatonin (pA(2) 10.6). Melatonin was also studied in superfused brain slices (from the guinea-pig) preincubated with [(3)H]noradrenaline. The electrically evoked tritium overflow in cerebrocortical, hippocampal and hypothalamic slices (representing quasi-physiological noradrenaline release) and in striatal slices (representing quasi-physiological dopamine release) was not affected by melatonin at a concentration that causes the maximum effect in retinal discs. In conclusion, dopamine release in the guinea-pig retina is inhibited via G(i)-PCRs including dopamine (D(2/3)), muscarinic and melatonin (MT(2)) receptors but not affected via any of the G(s)- or G(q)-PCRs under study. Unlike in the retina, melatonin fails to inhibit monoamine release in four brain regions of the guinea-pig.

Evidence for the involvement of two pathways in activation of extracellular signal-regulated kinase (Erk) and cell proliferation by Gi and Gq protein-coupled receptors in osteoblast-like cells.

The mechanisms by which Gi and Gq protein- coupled receptors mediate mitogenic signaling in osteoblast-like cells are unknown and were investigated in MC3T3-E1 cells using specific receptor agonists such as lysophosphatidic acid (LPA) and prostaglandin F2alpha (PGF2alpha). In contrast to their implication in epidermal growth factor (EGF) receptor tyrosine kinase signaling, the adaptor protein Shc, the Grb2/Sos complex, and the small G protein Ras were not involved in the activation of Erk induced by either LPA or PGF2alpha in MC3T3-E1 cells, suggesting that activation of Erk by Gi and Gq protein-coupled receptors is Ras independent in these cells. Using specific kinase inhibitors and kinetic analyses, we provide evidence for two distinct components in the activation of Erk by Gi and Gq protein-coupled receptors in MC3T3-E1 cells including an Src-like kinase-dependent pathway and a protein kinase C (PKC)-dependent mechanism. Functional analyses suggested that these two components are required for optimal DNA synthesis in response to LPA and PGF2alpha. These results suggest the implication of two pathways in the stimulation of Erk and cell replication by growth factors acting through Gi and Gq protein-coupled receptors in bone-forming cells.

VIP activates G(s) and G(i3) in rat alveolar macrophages and G(s) in HEK293 cells transfected with the human VPAC(1) receptor.

We have characterized vasoactive intestinal peptide (VIP) receptor/G-protein coupling in rat alveolar macrophage (AM) membranes and find that pertussis toxin treatment and antisera against G(alphai3) and G(alphas) reduce high-affinity (125)I-VIP binding, indicating that both G(alphas) and G(alphai3) couple to the VIP-receptor. The predominant VIP-receptor subtype in AM is VPAC(1) and we examined the G-protein interactions of the human VPAC(1) that had been transfected into HEK293 cells. VPAC(1) has a molecular mass of 56 kDa; GTP analogs reduced (125)I-VIP binding to this protein demonstrating that high-affinity binding of VIP to the receptor requires coupling to G-protein. Functional VIP/VPAC(1)/G-protein complexes were captured by covalent cross-linking and analyzed by Western blotting. The transfected human VPAC(1) receptor in HEK293 was found to be coupled to G(alphas) but not G(alphai) or G(alphaq). Furthermore, pertussis toxin treatment had no effect on VPAC(1)/G-protein coupling in these cells. These observations suggest that the G-proteins activated by VPAC(1) may be dependent upon species and cell type.

Sensitivity of orexin-A binding to phospholipase C inhibitors, neuropeptide Y, and secretin.

The binding of [(125)I] orexin-A (Ox-A) to particulates from Chinese hamster ovary (CHO) cells expressing the cloned orexin-A receptor, or from rat forebrain areas, was sensitive to blockers of phosphatidylinositol-specific phospholipase C (PtdIns-PLC) U-73122 and ET-18-OCH(3), little affected by phospholipase A(2) inhibitor quinacrine, and not sensitive to D609, a xanthate inhibitor of phosphatidylcholine-selective PLC. Interaction of the receptor with a PtdIns-PLC was further indicated by a large sensitivity of the binding to Ca(2+). Up to 50% of the binding was sensitive to the G-protein nucleotide site agonist GTP-gamma-S. Ligand attachment to the orexin-A receptor thus depends on an association with both PtdIns-PLC and G-protein alpha-subunits. In all paradigms examined, the binding of [(125)I]orexin-A was competed by human/rat neuropeptide Y (hNPY) and porcine secretin with a potency similar to orexin-A (IC(50) range 30-100 nM). The rank order of potency for NPY-related peptides was hNPY > porcine peptide YY (pPYY) > (Leu(31), Pro(34)) human PYY > human PYY(3-36) > hNPY free acid > human pancreatic polypeptide. Among secretin-related peptides, the rank order of potency was porcine secretin > or = orexin-A > human pituitary adenylate cyclase-activating peptide > orexin-B > porcine vasoactive intestinal peptide. Among opioid peptides, rat beta-endorphin and camel delta-endorphin were much less active than NPY and secretin, and two enkephalins were inactive at 1 microM. In view of high abundance of NPY in forebrain, the above cross-reactivity could indicate a significant contribution of NPY to signaling via orexin-A receptors.

Interleukin-8 and other agonists of Gi2 proteins: autocrine paracrine growth factors for human hematopoietic progenitors acting in synergy with colony stimulating factors.

We have reviewed the current knowledge on CXC chemokine interleukin-8 (IL-8) and human hematopoiesis, and more generally on agonists of heterotrimeric Gi2 proteins as regulators of human hematopoiesis. It appears that low doses of IL-8, a Gi2-agonist produced in an autocrine fashion by normal hematopoietic progenitors, mature blood cells and leukemic cells, promotes cell survival or/and proliferation in response to hematopoietic cytokines. More importantly, inactivation of the IL-8/Gi2 pathways inhibits CD34+ cell proliferation and colony formation. Similar positive effects on hematopoiesis of other, physiological or pathological, agonists of Gi2 proteins are discussed, as well as the molecular pathways involved and the consequences of activation of other G proteins (Gq, G16) by IL-8 and other Gi2-agonists.