Multiple G-protein involvement in parathyroid hormone regulation of acid production by osteoclasts

The involvement of multiple G-proteins in parathyroid hormone regulation of acid production was demonstrated in a highly enriched osteoclast population. Osteoclasts were isolated from the endosteum of 2.5 to 3-week-old chicken tibia using sequential enzymatic digestion. Single cell analysis of acid production was accomplished using microscope photometry and vital staining with acridine orange, a hydrogen ion concentration sensitive fluorescent dye. Lithium chloride, an uncoupler of G-proteins from their respective receptors, blocked parathyroid hormone stimulated production of acid. Cholera toxin, which permanently activates G_s-proteins, mimicked PTH stimulation. Pertussis toxin, which prevents receptor interaction with G_i- and G_o-proteins, blocked both 10 ⁸ M and 10 ¹¹ M PTH stimulated acid production, suggesting that the pertussis toxin-sensitive G-protein is utilized at both PTH concentrations. Immunoblots of osteoclast plasma membrane proteins, using a panel of antibodies generated against specific G-protein α subunits, revealed a 48 kDa G_sα, a 41 G_oα, a 34 kDa G_iα-3, and a unique 68 kDa Gα subunit, with the 41 kDa and 34 kDa bands being the most intense. Immunoblots of osteoblast plasma membrane proteins had a substantially different profile with the most intense bands being a G_sα (48 kDa) and a G_oα (36 and 38 kDa). The studies suggest the utilization of at least two different G-proteins in the parathyroid hormone regulation of acid formation by osteoclasts, a G_s and a pertussis toxin-sensitive G-protein (G_o and/or G_iα-3). J. Cell. Biochem. 64:161–170. © 1997 Wiley-Liss, Inc.     

Heterotrimeric G-protein and signal transduction in the nematode-trapping fungus Arthrobotrys dactyloides

The fungus Arthrobotrys dactyloides produces specialized constricting rings to trap and then consume nematodes. The signal transduction pathway involved in the nematode-trapping process was examined. Mastoparan, an activator of G-protein, had a stimulatory effect on the inflation of ring cells, whereas a G-protein inhibitor, pertussis toxin, prevented ring-cell expansion. The 40-kDa G_α of heterotrimeric G-proteins was specifically ADP-ribosylated by pertussis toxin. Using an antibody specific to the 35-kDa subunit G_β, we showed that immunogold-labeled G_β was more concentrated in ring cells than in the hyphae. In the absence of nematodes, the rings could be inflated by either pressurizing the culture in a syringe, raising intracellular Ca²⁺ concentrations, or adding warm water. We used these methods to reveal differences in responses to antagonists. The results support a model in which the pressure exerted by a nematode on the ring activates G-proteins in the ring cells. The activation leads to an increase in cytoplasmic Ca²⁺, activation of calmodulin, and finally the opening of water channels. The ring cells expand to constrict the ring and thus immobilize the nematode. Received: 13 April 2000 / Accepted: 22 June 2000     

Neuronal Platelet-Activating Factor Receptor Signal Transduction Involves a Pertussis Toxin-Sensitive G-Protein

In most nonneural systems, platelet-activating factor (PAF) receptor effects are mediated by G-proteins that are often pertussis toxin-sensitive. The activation of pertussis toxin-sensitive G-proteins linked to PAF receptors results in the mobilization of intracellular calcium, at least in part, through the second messenger inositol triphosphate. We have sought to determine if a pertussis toxin-sensitive G-protein is involved in the PAF receptor-mediated phenomena of growth cone collapse and of synaptic enhancement in primary neuronal culture. Using infrared differential interference contrast microscopy and patch-clamp recording techniques, pertussis toxin, but not the inactive B oligomer of the toxin, was found to block both the growth cone collapse and the enhanced synaptic release of excitatory transmitter induced by a nonhydrolyzable PAF receptor agonist, making it likely that G_o, G_q, or G_i is the G-protein transducer of PAF receptors in primary neurons. We believe that PAF acts directly on neuronal receptors, which are linked to pertussis toxin-sensitive G-proteins, on the tips of developing neurites, and on presynaptic nerve terminals, leading to growth cone collapse and enhanced synaptic release of transmitter.     

Pertussis toxin-sensitive G protein that supports vitellogenin uptake by promoting patency

Ovarian follicles of Hyalophora cecropia stopped accumulating [³⁵S]vitellogenin when incubated in pertussis toxin, a G_i protein inactivator. At a cellular level, the responses to pertussis toxin resembled those described earlier to cell-permeant analogs of cyclic AMP. They included accelerated ³⁶Cl^– exchange, ⁸⁶Rb⁺ uptake, and follicle cell swelling, which in turn resulted in a loss of epithelial patency. A 34% rise in follicular cAMP content accompanied these changes. In particulate fractions of follicle homogenates, pertussis toxin catalyzed the ADP-ribosylation of a polypeptide that resolved at 39 kDa in SDS-PAGE; rabbit antibodies to a C-terminal decapeptide common to 39 kDa mammalian G_iα-3 and G_oα were bound in immunoblots at this same location. The findings suggest that a pertussis toxin-sensitive Gα facilitates epithelial patency during vitellogenesis by suppressing cAMP levels. When follicles are released from this restraint, either experimentally with pertussis toxin or by progressing to the next phase in their normal program of development, cAMP levels rise and vitellogenesis terminates. Arch. Insect Biochem. Physiol. 39:36–45, 1998. © 1998 Wiley-Liss, Inc.     

Quartz crystal microbalance investigation of the interaction of bacterial toxins with ganglioside containing solid supported membranes

The binding of cholera toxin, tetanus toxin and pertussis toxin to ganglioside containing solid supported membranes has been investigated by quartz crystal microbalance measurements. The bilayers were prepared by fusion of phospholipid-vesicles on a hydrophobic monolayer of octanethiol chemisorbed on one gold electrode placed on the 5 MHz AT-cut quartz crystal. The ability of the gangliosides G_M1, G_M3, G_D1a, G_D1b, G_T1b and asialo-G_M1 to act as suitable receptors for the different toxins was tested by measuring the changes of quartz resonance frequencies. To obtain the binding constants of each ligand-receptor-couple Langmuir-isotherms were successfully fitted to the experimental adsorption isotherms. Cholera toxin shows a high affinity for G_M1 (K_a = 1.8 ⋅ 10⁸M^–1), a lower one for asialo-G_M1 (K_a = 1.0 ⋅ 10⁷ M^–1) and no affinity for G_M3. The C-fragment of tetanus toxin binds to ganglioside G_D1a, G_D1b and G_T1b containing membranes with similar affinity (K_a∼10⁶ M^–1), while no binding was observed with G_M3. Pertussis toxin binds to membranes containing the ganglioside G_D1a with a binding constant of K_a = 1.6 ⋅ 10⁶ M^–1, but only if large amounts (40 mol%) of G_D1a are present. The maximum frequency shift caused by the protein adsorption depends strongly on the molecular structure of the receptor. This is clearly demonstrated by an observed maximum frequency decrease of 99 Hz for the adsorption of the C-fragment of tetanus toxin to G_D1b. In contrast to this large frequency decrease, which was unexpectedly high with respect to Sauerbrey's equation, implying pure mass loading, a maximum shift of only 28 Hz was detected after adsorption of the C-fragment of tetanus toxin to G_D1a. Received: 14 January 1997 / Accepted: 15 April 1997     

Role of MMP-2 in PKCδ-mediated inhibition of Na+ dependent Ca2+ uptake in microsomes of pulmonary smooth muscle: Involvement of a pertussis toxin sensitive protein

Treatment of bovine pulmonary artery smooth muscle with the O₂^•− generating system hypoxanthine plus xanthine oxidase stimulated MMP-2 activity and PKC activity; and inhibited Na⁺ dependent Ca²⁺ uptake in the microsomes. Pretreatment of the smooth muscle with SOD (the O₂^•− scavenger) and TIMP-2 (MMP-2 inhibitor) prevented the increase in MMP-2 activity and PKC activity, and reversed the inhibition of Na⁺ dependent Ca²⁺ uptake in the microsomes. Pretreatment with calphostin C (a general PKC inhibitor) and rottlerin (a PKCδ inhibitor) prevented the increase in PKC activity and reversed O₂^•− caused inhibition of Na⁺ dependent Ca²⁺ uptake without causing any change in MMP-2 activity in the microsomes of the smooth muscle. Treatment of the smooth muscle with the O₂^•− generating system revealed, respectively, 36 kDa RACK-1 and 78 kDa PKCδ immunoreactive protein profile along with an additional 38 kDa immunoreactive fragment in the microsomes. The 38 kDa band appeared to be the proteolytic fragment of the 78 kDa PKCδ since pretreatment with TIMP-2 abolished the increase in the 38 kDa immunoreactive fragment. Co-immunoprecipitation of PKCδ and RACK-1 demonstrated O₂^•− dependent increase in PKCδ-RACK-1 interaction in the microsomes. Immunoblot assay elicited an immunoreactive band of 41 kDa G_iα in the microsomes. Treatment of the smooth muscle tissue with the O₂^•− generating system causes phosphorylation of G_iα in the microsomes and pretreatment with TIMP-2 and rottlerin prevented the phosphorylation. Pretreatment of the smooth muscle tissue with pertussis toxin reversed O₂^•− caused inhibition of Na⁺ dependent Ca²⁺ uptake without affecting the protease activity and PKC activity in the microsomes. We suggest the existence of a pertussis toxin sensitive G protein mediated mechanism for inhibition of Na⁺ dependent Ca²⁺ uptake in microsomes of bovine pulmonary artery smooth muscle under O₂^•− triggered condition, which is regulated by PKCδ dependent phosphorylation and sensitive to TIMP-2 for its inhibition. (Mol Cell Biochem xxx: 107–117, 2005)     

Distribution of the α-subunit of the Guanine Nucleotide-binding Protein Gi2 and its Comparison to Gαo

Abstract

Site specific antisera against a synthetic peptide corresponding to the sequence 3–17 of Gαi2 have been raised and the specificity examined using purified homogeneous G_o, G_i2 and G_i containing a 41 kDa α-subunit. The distribution of Gα_i2 was investigated in plasma membranes from different tissues and cells and compared to the distribution of Gα_o and other pertussis toxin sensitive Gα. Considerable amounts of Gα_io were found in endocrine tissue especially in membranes from the adrenal and thyroid, in leucocytes and platelets where it constitutes the major, if not only, pertussis toxin-sensitive Gα, as well as in some cell lines (C6, NG 108–15, S49 cyc^?); erythrocytes contained a 41 kDa Gα_i which was different from Gα_o. Gα_o was present abundantly in nervous tissue, adrenal medulla and cortex but also found in low amounts in other membranes except for lung, liver and blood cells. Subcellular fractionaltion of cardiac ventricular muscle demonstrated the presence of Gα_o and low amounts of Gα_i2 in sarcolemma, but only 41kDa Gα_i was present in sarcoplasmic reticulum. The importance of the distinct distribution in terms of signal transduction is discussed.     

Pertussis-toxin insensitive enkephalin inhibition of adenylyl cyclase in lacrimal acinar cells

The mechanism by which the inhibitory effect of d-ala²-met-enkephalinamide (DALA) on lacrimal acinar adenylyl cyclase is exerted was assessed in membrane preparations by a cAMP protein binding assay. Inhibition by the analogue was GTP-dependent with a significant enhancement of the inhibitory effect by GTP. While pretreatment of membranes with either cholera or pertussis toxin resulted in stimulation of adenylyl cyclase activity, modification of the G_iα subunit by pertussis-toxin catalyzed ADP-ribosylation did not effect the hormonal inhibition of adenylyl cyclase. Incubation of membranes with manganese, however, prevented the inhibitory action of DALA in addition to enhancing basal and forskolin-stimulated adenylyl cyclase activity. The results suggest that the inhibitory effect of DALA in lacrimal acinar cells is exerted via a mechanism other than pertussis-toxin sensitive coupling of the receptor to adenylyl cyclase through G_i. The mechanism may be effected through a pertussis-toxin insensitive G protein, through an interaction with G_i that is pertussis-toxin insensitive, or through an interaction with the catalytic subunit of adenylyl cyclase.     

Adenosine Release and Uptake in Cerebellar Granule Neurons Both Occur via an Equilibrative Nucleoside Carrier that Is Modulated by G Proteins

Abstract: There is debate about the mechanisms mediating adenosine release from neurons. In this study, the release of adenosine evoked by depolarizing cultured cerebellar granule neurons with 50 mM K⁺ was inhibited by 49 ± 7% in Ca²⁺-free medium. The remaining release was blocked by dipyridamole (IC₅₀ = 6.4 × 10^?8M) and nitrobenzylthioinosine (IC₅₀ = 3.6 × 10^?8M), inhibitors of adenosine uptake. Ca²⁺-dependent release was reduced by 78 ± 9% following a 21-h pretreatment of the cells with pertussis toxin, which ADP-ribosylates G_i/G_o G proteins, thereby preventing their dissociation. The nucleoside transporter-mediated component of K⁺-induced adenosine release also was inhibited by 62 ± 8% by pertussis toxin and was potentiated by 78 ± 11% following cholera toxin treatment, which permanently activates G_s. Uptake of [³H]adenosine into cultured cerebellar granule neurons over a 10-min period was not dependent on extracellular Na⁺ but was reduced by dipyridamole (IC₅₀ = 3.2 × 10^?8M) and nitrobenzylthioinosine (IC₅₀ = 2.6 × 10^?8M). Thus, adenosine uptake likely occurs via the same transporter mediating Ca²⁺-independent adenosine release. Adenosine uptake was potentiated by cholera toxin pretreatment (152 ± 15% of control), but pertussis toxin had no statistically significant effect. It is possible that G_s, G_i/G_o, or free Gβγ dimer modulate the equilibrative, inhibitor-sensitive nucleoside carrier to enhance adenosine transport.     

Stimulation and conformational change of G<Subscript>o</Subscript>α induced by GAP-43

GAP-43 and G_o are peripheral membrane proteins enriched in neuronal growth cone. GAP-43 was highly purified from bovine cerebral cortex and myristoylated G_oα was highly purified from Escherichia coli cotransformed with pQE60 G_oα and pBB131 (NMT). GAP-43 stimulated GTPγS binding to G_oα and the stimulation effect was dependent on concentration of GAP-43. Protein-protein binding experiments using CaM-Sepharose affinity media revealed that G_oα GDP bound GAP-43 directly to form intermolecular complex. This interaction induced conformational change of G_oα. In the presence of GAP-43, fluorescence spectrum of G_oα GDP blue shifted 4 nm; fluorescence intensity increased 35.3% and apparent quenching constant (Ksv) increased from (1.1 ±0.22) ×10⁵ to (4.1±0.43) × 10⁵ (M⁻¹). However, no obvious changes of fluorescence spectra of G_oα GTPγS were observed in the absence or presence of GAP-43. Our results indicated that GAP-43 induced conformational change of G_oα GDP so as to accelerate GDP release and subsequent GTPγS binding, which activates G proteins to trigger signal transduction and amplification. These results provided insights into understanding the function of G proteins in coupling between receptors and effectors and the key role of GDP/GTP exchange mode in GTPase cycle.     

WNT-5A stimulates the GDP/GTP exchange at pertussis toxin-sensitive heterotrimeric G proteins

The lipoglycoproteins of the WNT family act on seven transmembrane-spanning Class Frizzled receptors. Here, we show that WNT-5A evokes a proliferative response in a mouse microglia-like cell line (N13), which is sensitive to pertussis toxin, thus implicating the involvement of heterotrimeric G proteins of the G_i/o family. We continue to show that WNT-5A stimulation of N13 membranes and permeabilized cells evokes the exchange of GDP for GTP of pertussis toxin-sensitive G proteins employing [γ-³⁵S]GTP assay and activity state-specific antibodies to GTP-bound G_i proteins. Our functional analysis of the PTX-sensitivity of WNT-induced G protein activation and PCR analysis of G protein and FZD expression patterns suggest that WNT-5A stimulation leads to the activation of G_i2/3 proteins in N13 cells possibly mediated by FZD₅, the predominant FZD expressed. In summary, we provide for the first time molecular proof that WNT-5A stimulation results in the activation of heterotrimeric G_i2/3 proteins in mammalian cells with physiological protein stochiometry.     

Pertussis toxin-sensitive G-proteins inhibit fibroblast growth factor-induced signaling in pancreatic acini

Signal transduction of fibroblast growth factor (FGF) receptors is known to involve tyrosine phosphorylation of several substrates, including Grb2, phospholipase C-γ, and phosphatidylinositol 3-kinase, whereas the role of G-proteins in FGF receptor signaling is controversial. In the present study we investigated the role of G-proteins in FGF receptor signaling in rat pancreatic acini. Immunological analysis revealed the presence of FGF receptor and phospholipase C-γ1 in rat pancreatic acini. Both basic fibroblast growth factor (FGF-2) and guanosine 5′-(γ-O-thio)triphosphate (GTPγS) caused an increase in inositol 1,4,5-trisphosphate (1,4,5-IP₃) production and amylase release. Combined stimulation of the acini with GTPγS and FGF-2 led to a decrease of these responses as compared to the effect of the single substances. When pancreatic acini were preincubated with FGF-2 (1 nM) or vehicle (water) ADP-ribosylation of the α-subunit of G_i-type G-proteins by pertussis toxin was reduced in membranes prepared from FGF-2 pretreated acini as compared to control acini, suggesting functional interaction of FGF receptors with G_i-proteins. Pretreatment of acini with pertussis toxin which inhibits G_i-type G-proteins abolished the inhibitory effect of GTPγS on FGF-induced 1,4,5-IP₃ production and amylase release, whereas the stimulatory effects of FGF-2 and GTPγS on these parameters remained unchanged. In conclusion, these results show communication of FGF receptors and G_i-type G-proteins and that G_i-type G-proteins exert an inhibitory influence on FGF-induced activation of phosphoinositide-specific phospholipase C in pancreatic acinar cells. © 1996 Wiley-Liss, Inc.     

Apical Heterotrimeric G-proteins Activate CFTR in the Native Sweat Duct

Other than the fact that the cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channel can be activated by cAMP dependent kinase (PKA), little is known about the signal transduction pathways regulating CFTR. Since G-proteins play a principal role in signal transduction regulating several ion channels [4, 5, 9], we sought to test whether G-proteins control CFTR Cl⁻ conductance (CFTR G _Cl ) in the native sweat duct (SD). We permeabilized the basolateral membrane with α-toxin so as to manipulate cytosolic nucleotides. We activated G-proteins and monitored CFTR G _Cl activity as described earlier [20, 23, 25]. We now show that activating G-proteins with GTP-γ-S (100 μm) also activates CFTR G _Cl in the presence of 5 mm ATP alone (without exogenous cAMP). GTP-γ-S increased CFTR G _Cl by 44 ± 20 mS/cm² (mean ±se; n= 7). GDP (10 mm) inhibited G-protein activation of CFTR G _Cl even in the presence of GTP-γ-S. The heterotrimeric G-protein activator (AlF₄ ⁻) in the cytoplasmic bath activated CFTR G _Cl (increased by 51.5 ± 9.4 mS/cm² in the presence of 5 mm ATP without cAMP, n= 6), the magnitude of which was similar to that induced by GTP-γ-S. Employing immunocytochemical-labeling techniques, we localized Gαs, Gαi, Gαq, and Gβ at the apical membranes of the sweat duct. Further, we showed that the mutant CFTR G _Cl in ducts from cystic fibrosis (CF) subjects could be partially activated by G-proteins. The magnitude of mutant CFTR G _Cl activation by G-proteins was smaller as compared to non-CF ducts but comparable to that induced by cAMP in CF ducts. We conclude that heterotrimeric G-proteins are present in the apical membrane of the native human sweat duct which may help regulate salt absorption by controlling CFTR G _Cl activity. Received: 9 June 2000/Revised: 5 October 2000     

Purification and characterization of G proteins from human brain: modification of GTPase activity upon phosphorylation

Summary Three G proteins from human brain membranes were purified to near homogeneity by conventional techniques including preparative electrophoresis. These G proteins were characterized by their ability to bind GTP, GDP and GTP analogs. Two of these proteins have molecular weights of 50,000 (G₅₀) and 36,000 (G₃₆), as determined on SDS-gels. G₃₆ was ADP-ribosylated by pertussis toxin. Thus, G₅₀ could represent a G_sα subunit, whereas G₃₆ could be G_iα or G_oα. G₅₀ was phosphorylated by cAMP dependent protein kinase and protein kinase C. G₃₆ was phosphorylated by a protein kinase independent of calcium and phospholipid, a proteolytic product of protein kinase C, analogous to protein kinase M. Phosphorylation of G₃₆ by this protein kinase induced a dramatic decrease in its GTPase activity. The third G protein, of molecular weight 22,000 probably belongs to the group of monomeric G proteins possessing functional similarities withras gene products. The regulation of G proteins involving calcium-dependent and independent pathways is delineated.     

Adenosine A1 Agonists and the Ca2+ Channel Agonist Bay K 8644 Produce a Synergistic Stimulation of the GTPase Activity of Go in Rat Frontal Cortical Membranes

Abstract: The identity and role of G proteins in coupling adenosine receptors to effectors have been studied to a limited degree. We have identified the G proteins whose GTPase activity is stimulated by adenosine receptor agonists in neuronal membranes. (R)-Phenylisopropyladenosine, 2-chloroadenosine, and N-ethylcarboxamideadenosine produced a concentration-dependent stimulation of GTPase. At 10^?5M, the increase above basal GTPase in frontal cortex was 25 ± 4, 20 ± 3, and 8 ± 1%, respectively, and in the cerebellum 55 ± 2, 41 ± 4, and 22 ± 2%, respectively. The effects of (R)-phenylisopropyladenosine and 2-chloroadenosine were inhibited by (1) A₁ antagonists (76–96% reduction), (2) pretreatment with pertussis toxin (90–100% reduction), and (3) antibodies raised against the α-subunit of G_i and G_o (55–57% reduction by each), suggesting that A₁ receptors interact equally with G_i and G_o. (R)-Phenylisopropyladenosine increased the binding of a nonhydrolyzable analogue of GTP to membranes in a pertussis toxin-sensitive manner, indicative of activation of G_i or G_o. Previously, (±)-Bay K 8644 enhanced GTP hydrolysis by G_o but not G_i. Now we report a profound synergistic stimulation of GTPase in the presence of (R)-phenylisopropyladenosine and (±)-Bay K 8644 (10^?7 to 10^?5M). (±)-Bay K 8644 had no effect on nucleotide exchange and, thus, cannot activate G_o. It appears that a positive cooperative stimulation of G_o occurs when it is first activated by A₁ receptors and subsequently interacts with the L-type Ca²⁺ channel.     

Cholera toxin and pertussis toxin substrates and endogenous ADP-ribosyltransferase activity in Drosophila melanogaster

Cholera toxin- and pertussis toxin-catalyzed ADP-ribosylation were used to identify and localize G protein substrates in Drosophila melanogaster and in Manduca sexta. Cholera toxin catalyzes ADP-ribosylation of 37 kDa and 50 kDa polypeptides, but these polypeptides are also substrates for an ADP-ribosyltransferase (EC 2.4.2.30) activity endogenous to the Drosophila extracts. Pertussis toxin modifies 37 kDa and 39 kDa polypeptides in Drosophila homogenates. The pattern of proteolysis of the 39 kDa pertussis toxin substrate is similar to that of mammalian G_o and is influenced by guanyl nucleotide binding. The 39 kDa G_o-like Drosophila and Manduca pertussis toxin substrates are found primarily in neural tissues. These studies provide further evidence that G proteins are present in Drosophila and that this organism can therefore be used to investigate the physiological roles of these enzymes using advanced genetic manipulations.     

Sphingosylphosphorylcholine and sphingosinew-1-phosphate mobilize cytosolic calcium through different mechanisms in human airway epithelial cells

The sphingosine derivatives sphingosylphosphorylcholine (SPC) and sphingosine-1-phosphate (S1P) caused a similar elevation of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in an immortalized airway epithelial cell line (CFNP9o⁻) incubated in Ca²⁺-free medium. The maximal effect was obtained with 2 μM SPC and 0.1 μM S1 P and was sensitive to pre-incubation with pertussis toxin, indicating the involvement of a G_i/G_o type of G protein. In Ca²⁺ containing medium, [Ca²⁺]_i elevation by SPC was significantly higher than that by S1P, due to the fact that SPC was able to stimulate Mn²⁺ entry, whereas S1P was ineffective. SPC, but not S1P, caused a dose-dependent production of total inositol phosphates. Conversely, S1P, but not SPC, increased the level of phosphatidic acid. These findings suggest the presence of two distinct receptors, specific for SPC and S1P, respectively. Depletion of intracellular Ca²⁺ stores by SPC makes cells unable to respond to a subsequent addition of S1P. Conversely, cells do respond to SPC after a challenge with S1P, suggesting that the two receptors likely share one or more intracellular signalling component(s).     

Effect of pertussis toxin on the phosphodiesteratic cleavage of the polyphosphoinositides by guanosine 5′-O-thiotriphosphate and thrombin in permeabilized human platelets

It has recently been shown in this laboratory that permeabilization of human platelets with 15–25 μm/ml saponin allows ADP-ribosylation by pertussis toxin of the α_i-subunit of G_i(N_i), a guanine nucleotide-binding regulatory protein. The same assay conditions have been used to determine phospholipase C in permeabilized platelets. Guanosine 5′-O-thiophosphate- (GTP[γS]-) activated phospholipase C in permeabilized platelets whose inositol phospholipids were prelabeled with [³H]inositol. Phospholipase C activity was measured by [³H]polyphosphoinositide decreases and formation of [³Hinositol bisphosphate and [³H]inositol trisphosphate. Prostacyclin, cyclic AMP or pretreatment of permeabilized platelets with pertussis toxin did not alter this effect under conditions in which the α_i-subunit was effectively ADP-ribosylated by pertussis toxin. This information indicated that ADP-ribosylation of G_i-protein was not directly related to activation or inhibition of platelet phospholipase C by GTP[γS]. Thrombin also activated phospholipase C in permeabilized platelets and, surprisingly, this action was enhanced by pertussis toxin pretreatment. This indicated that ADP-ribosylation of G_i-protein facilitates the action of thrombin on phospholipase C.     

Role of cysteine 341 and arginine 348 of GLP-1 receptor in G-protein coupling

We have demonstrated the ability of peptides derived from the third intracellular loop of GLP-1 receptor to differently modulate activity of four different types of G-proteins overexpressed in sf9 cells. In this respect, the involvement of Cys³⁴¹ in inhibition of G_s and Cys³⁴¹ in activation of G_s and in inhibition of G_i1, G_o, and G₁₁, respectively, indicates their potential role in discrimination between different types of G-proteins. Moreover, these two amino acids from the third intracellular loop might represent an important novel targets for covalent modification by downstream regulators in signaling through GLP-1 receptor.     

Regulation of cellular signals by G-proteins

Extracellular signals are transduced across the cell by the cell surface receptors, with the aid of G-proteins, which act at a critical point of signal transduction and cellular regulation. Structurally, G-proteins are heterotrimeric consisting α, β and γ subunits but in functionally active state they dissociate into α subunit coupled to GTP and as βγ dimer. G-proteins can be broadly divided into two classes based on their sensitivity to pertussis toxin and cholera toxin. Existence of various forms of each of the subunit allows molecular diversity in the subunit species of G-proteins. These subunits interact with a wide range of receptors and effectors, facilitated by post translational modification of their subunits. Different types of G-proteins mediate several signalling events in different parts of the body. This review summarizes the features of (i) structural and functional heterogenity among different subunits of G-proteins, (ii) interaction of G-proteins and their subunits with effectors with specific cases of G-protein mediated signalling in olfaction, phototransduction in the retina, ras andras related transduction and (iii) disease conditions associated with malfunctioning of G-proteins.