scholarly journals Regulation of bombesin-stimulated inositol 1,4,5-trisphosphate generation in Swiss 3T3 fibroblasts by a guanine-nucleotide-binding protein

1990 ◽  
Vol 268 (3) ◽  
pp. 605-610 ◽  
Author(s):  
R Plevin ◽  
S Palmer ◽  
S D Gardner ◽  
M J O Wakelam
Keyword(s):  
G Protein ◽  
Protein Interactions ◽  
Inositol Phosphate ◽  
Early Time ◽  
Lag Time ◽  
Maximal Response ◽  
Guanine Nucleotide Binding Protein ◽  
3T3 Fibroblasts ◽  
Swiss 3T3 ◽  
Guanine Nucleotide Binding

The stimulation of inositol phosphate generation by bombesin and GTP analogues was studied in Swiss 3T3 cells permeabilized by electroporation. Bombesin-stimulated inositol phosphate generation is potentiated by guanosine 5′-[gamma-thio]triphosphate (GTP[S]) and inhibited by guanosine 5′-[beta-thio]diphosphate at all peptide concentrations tested, with no change in the EC50 value (concn. giving half-maximal response) for the agonist. Kinetic analysis showed that, although bombesin-stimulated [3H]InsP3 generation in [3H]inositol-labelled cells was rapid (maximal by 5-10 s), the response to GTP[S] alone displayed a distinct lag time of 20-30 s. This lag time was significantly decreased by the addition of bombesin, suggesting that in this system agonist-stimulated GTP/GDP exchange occurs. In addition, bombesin-stimulated generation of Ins(1,4,5)P3 mass at 10 s was enhanced by GTP[S] in the absence of a nucleotide response alone, a result consistent with this proposal. Pretreatment of the cells with phorbol 12-myristate 13-acetate (PMA) resulted in a dose-dependent inhibition of bombesin-, but not GTP[S]-, stimulated inositol phosphate generation. Furthermore, although PMA pretreatment did not affect the lag time for InsP3 formation in response to GTP[S] alone, the degree of synergy between bombesin and the nucleotide was severely decreased at early time points. The results therefore demonstrate that the high-affinity bombesin receptor is coupled via a G-protein to phospholipase C in a manner consistent with a general model for receptor-G-protein interactions and that this coupling is sensitive to phosphorylation by protein kinase C.

Role of G proteins and KCa channels in the muscarinic and beta-adrenergic regulation of airway smooth muscle

1995 ◽  
Vol 268 (2) ◽  
pp. L221-L229 ◽  
Author(s):  
H. Kume ◽  
K. Mikawa ◽  
K. Takagi ◽  
M. I. Kotlikoff
Keyword(s):  
Smooth Muscle ◽  
Adenylyl Cyclase ◽  
G Protein ◽  
Muscarinic Receptor ◽  
G Proteins ◽  
Isometric Tension ◽  
Guanine Nucleotide Binding Protein ◽  
Kca Channels ◽  
Stimulatory G Protein ◽  
Guanine Nucleotide Binding

We have examined the functional consequences of G protein coupling to calcium-activated potassium (KCa) channels using isometric tension records from guinea pig tracheal smooth muscle. After incubation with 1 microgram/ml pertussis toxin (PTX) for 6 h, the contraction response to 1 microM methacholine (MCh) was suppressed by 31.7 +/- 5.0% (n = 10). Similarly, the contraction was inhibited by 29.1 +/- 5.0% (n = 6) after application of 0.1 microM AF-DX 116, an M2-selective muscarinic receptor antagonist. Cholera toxin (CTX, 2.0 micrograms/ml for 6 h), which activates the stimulatory G protein of adenylyl cyclase (Gs), also suppressed contraction by 43.9 +/- 3.3% (n = 11). The inhibitory effects of PTX, AF-DX 116, or CTX were reversed in the presence of 100 nM charybdotoxin (ChTX), a selective KCa channel inhibitor. These findings suggest that disruption of inhibitory coupling between muscarinic receptor and KCa channels mediated by PTX-sensitive G proteins, or KCa channel activation induced by Gs/adenylyl cyclase-linked processes, antagonizes muscarinic contraction. The isoproterenol concentration-inhibition curves for precontracted trachea (1 microM MCh) were shifted to the left after perfusion with PTX or AF-DX 116, and the leftward shift of the curve was blocked by ChTX. Thus direct or indirect regulation of KCa channels mediated by the inhibitory guanine nucleotide binding protein (Gi) and Gs may play a functionally important role in the mechanical antagonism by the two receptor agonists.

Guanine Nucleotide Binding Protein (G Protein), Gamma

2012 ◽  
pp. 831-831
Author(s):  
Thomas E. Meigs ◽  
Alex Lyakhovich ◽  
Hoon Shim ◽  
Ching-Kang Chen ◽  
Denis J. Dupré ◽  
...  
Keyword(s):  
G Protein ◽  
Binding Protein ◽  
Nucleotide Binding ◽  
Protein G ◽  
Guanine Nucleotide ◽  
Guanine Nucleotide Binding Protein ◽  
Guanine Nucleotide Binding ◽  
Nucleotide Binding Protein

scholarly journals Inhibition by pertussis toxin of the activation of Na+-dependent uridine transport in dimethyl-sulphoxide-induced HL-60 leukaemia cells

1991 ◽  
Vol 280 (2) ◽  
pp. 515-519 ◽  
Author(s):  
J A Sokoloski ◽  
A C Sartorelli ◽  
R E Handschumacher ◽  
C W Lee
Keyword(s):  
Cholera Toxin ◽  
G Protein ◽  
Pertussis Toxin ◽  
Nucleotide Binding ◽  
Dimethyl Sulphoxide ◽  
Protein G ◽  
Guanine Nucleotide ◽  
Guanine Nucleotide Binding Protein ◽  
Guanine Nucleotide Binding ◽  
Dependent Transport

The effects of pertussis toxin on the Na(+)-dependent transport of uridine were studied in HL-60 leukaemia cells induced to differentiate along the granulocytic or monocytic pathways by dimethyl sulphoxide (DMSO) or phorbol 12-myristate 13-acetate (PMA) respectively. Pertussis toxin at 50 ng/ml completely inhibited the activation of Na(+)-dependent uridine transport and consequently prevented the formation of intracellular pools of free uridine which occurs in HL-60 cells induced to differentiate by DMSO. The inhibition of Na(+)-dependent uridine transport by pertussis toxin in cells exposed to DMSO was associated with a 14-fold decrease in affinity, with no change in Vmax. Pertussis toxin, however, had no effect on Na(+)-dependent uridine transport in PMA-induced HL-60 cells. Furthermore, 500 ng of cholera toxin/ml had no effect on the Na(+)-dependent uptake of uridine in DMSO-treated HL-60 cells. These results suggest that the activation of the Na(+)-dependent transport of uridine in HL-60 cells induced to differentiate along the granulocytic pathway by DMSO is coupled to a pertussis-toxin-sensitive guanine-nucleotide binding protein (G-protein).

scholarly journals Crystal structure of rhodopsin in complex with a mini-Gosheds light on the principles of G protein selectivity

2018 ◽  
Vol 4 (9) ◽  
pp. eaat7052 ◽  
Author(s):  
Ching-Ju Tsai ◽  
Filip Pamula ◽  
Rony Nehmé ◽  
Jonas Mühle ◽  
Tobias Weinert ◽  
...  
Keyword(s):  
Crystal Structure ◽  
G Protein ◽  
Maximum Efficiency ◽  
Guanine Nucleotide Binding Protein ◽  
Precise Position ◽  
Protein Activation ◽  
Signaling Complex ◽  
G Protein Activation ◽  
Clinical Drugs ◽  
Guanine Nucleotide Binding

Selective coupling of G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptors (GPCRs) to specific Gα-protein subtypes is critical to transform extracellular signals, carried by natural ligands and clinical drugs, into cellular responses. At the center of this transduction event lies the formation of a signaling complex between the receptor and G protein. We report the crystal structure of light-sensitive GPCR rhodopsin bound to an engineered mini-Goprotein. The conformation of the receptor is identical to all previous structures of active rhodopsin, including the complex with arrestin. Thus, rhodopsin seems to adopt predominantly one thermodynamically stable active conformation, effectively acting like a “structural switch,” allowing for maximum efficiency in the visual system. Furthermore, our analysis of the well-defined GPCR–G protein interface suggests that the precise position of the carboxyl-terminal “hook-like” element of the G protein (its four last residues) relative to the TM7/helix 8 (H8) joint of the receptor is a significant determinant in selective G protein activation.

scholarly journals Evidence that activation of a common G-protein by receptors for leukotriene B4 and N-formylmethionyl-leucyl-phenylalanine in HL-60 cells occurs by different mechanisms

1989 ◽  
Vol 260 (2) ◽  
pp. 427-434 ◽  
Author(s):  
K R McLeish ◽  
P Gierschik ◽  
T Schepers ◽  
D Sidiropoulos ◽  
K H Jakobs
Keyword(s):  
Cholera Toxin ◽  
G Protein ◽  
Leukotriene B4 ◽  
Alpha Subunit ◽  
Gtpase Activity ◽  
Protein Activity ◽  
Guanine Nucleotide Binding Protein ◽  
Gtp Hydrolysis ◽  
Fmlp Receptor ◽  
Guanine Nucleotide Binding

Differentiated HL-60 cells were found to respond to the chemoattractants leukotriene B4 (LTB4) and N-formylmethionyl-leucyl-phenylalanine (FMLP), in a manner similar to neutrophils. Membranes of myeloid differentiated HL-60 cells were used (a) to examine the ability of LTB4 receptors to interact with a guanine-nucleotide-binding protein (G-protein), and (b) to compare this G-protein with that which is coupled to the FMLP receptor. LTB4 stimulated a dose-dependent increase in GTP hydrolysis and guanosine 5′-[gamma-thio]triphosphate (GTP[S]) binding, demonstrating that LTB4 receptors on HL-60 cells are coupled to a G-protein. Both pertussis toxin and cholera toxin inhibited stimulation of GTPase activity and GTP[S] binding by either LTB4 or FMLP, indicating that both receptors are coupled to a G-protein containing a 40 kDa alpha-subunit. That the two receptors share a common G-protein was shown by FMLP enhancement of cholera-toxin-induced inhibition of GTPase activity stimulated by either FMLP or LTB4. However, LTB4 did not enhance cholera-toxin-induced inhibition of GTPase activity, suggesting that the receptors interacted differently with this G-protein. This difference was confirmed by showing that FMLP, but not LTB4, stimulated receptor-specific [32P]ADP-ribosylation of the 40 kDa alpha-subunit. Concentrations of LTB4 and FMLP which produced maximal responses produced enhanced stimulation in both assays. This additive effect was not abolished by inactivation of up to 80% of G-protein activity by N-ethylmaleimide or cholera toxin. We conclude that LTB4 and FMLP receptors in HL-60 cells are coupled to a common G-protein. The receptor-G-protein interaction is different for the two receptors, and G-proteins not coupled to both receptors may account for the additive response.

scholarly journals Molecular cloning and primary structure of squid (Loligo forbesi) rhodopsin, a phospholipase C-directed G-protein-linked receptor

1991 ◽  
Vol 274 (1) ◽  
pp. 35-40 ◽  
Author(s):  
M D Hall ◽  
M A Hoon ◽  
N J P Ryba ◽  
J D D Pottinger ◽  
J N Keen ◽  
...  
Keyword(s):  
G Protein ◽  
Primary Structure ◽  
Protein G ◽  
Guanine Nucleotide ◽  
Cdna Sequencing ◽  
Functional Importance ◽  
Guanine Nucleotide Binding Protein ◽  
C Terminus ◽  
Loligo Forbesi ◽  
Guanine Nucleotide Binding

The sequence of squid (Loligo forbesi) rhodopsin was determined by protein and cDNA sequencing. The protein has close similarity to octopus rhodopsin, having an N-terminal region (residues 1-340) which resembles other guanine-nucleotide-binding protein (G-protein)-linked receptors and a repetitive proline-rich C-terminus (residues 340-452). Comparison of the sequence of squid rhodopsin with those of other members of the G-protein-linked receptor superfamily reveals features which we predict to have both structural and functional importance.

scholarly journals Multiple sources of sn-1,2-diacylglycerol in platelet-derived-growth-factor-stimulated Swiss 3T3 fibroblasts. Evidence for activation of phosphoinositidase C and phosphatidylcholine-specific phospholipase D

1991 ◽  
Vol 279 (2) ◽  
pp. 559-565 ◽  
Author(s):  
R Plevin ◽  
S J Cook ◽  
S Palmer ◽  
M J O Wakelam
Keyword(s):  
Growth Factor ◽  
Phospholipase D ◽  
Lag Time ◽  
Water Soluble ◽  
Platelet Derived Growth Factor ◽  
Second Phase ◽  
Multiple Sources ◽  
3T3 Fibroblasts ◽  
Swiss 3T3 ◽  
Swiss 3T3 Fibroblasts

Platelet-derived growth factor (PDGF) stimulated sn-1,2-diacylglycerol (DAG) mass formation in Swiss 3T3 fibroblasts with a lag time of some 30 s. The response was biphasic, with the second phase being sustained over time. PDGF also stimulated the formation of Ins(1,4,5)P3 with a similar lag time to the DAG response, suggesting that DAG is derived from PtdIns(4,5)P2 hydrolysis at this time point. PDGF-stimulated phosphatidylcholine (PtdCho) hydrolysis in Swiss 3T3 fibroblasts, as measured by the formation of water-soluble choline metabolites and phosphatidylbutanol (PtdBut) accumulation, was by a phospholipase D (PLD)-catalysed pathway which was kinetically downstream of initial PtdIns(4,5)P2 hydrolysis. Accumulation of PtdBut increased up to 15 min, suggesting that PLD activity is not rapidly densitized in response to PDGF. The kinetics of PtdCho hydrolysis closely paralleled the second phase of DAG formation, strongly suggesting that during prolonged stimulation periods PtdCho is a major source of DAG in these cells. However, since PtdIns(4,5)P2 breakdown was also prolonged, PDGF-stimulated DAG may be derived from both phospholipids. Down-regulation of protein kinase C (PKC), by pre-treatment with phorbol 12-myristate 13-acetate, abolished both [3H]choline and [3H]PtdBut formation, suggesting that PLD-catalysed PtdCho hydrolysis may be dependent on PKC activation, supporting its dependence on prior PtdIns(4,5)P2 hydrolysis.

Metalloprotease cleavage of the N terminus of the orphan G protein–coupled receptor GPR37L1 reduces its constitutive activity

2016 ◽  
Vol 9 (423) ◽  
pp. ra36-ra36 ◽  
Author(s):  
James L. J. Coleman ◽  
Tony Ngo ◽  
Johannes Schmidt ◽  
Nadine Mrad ◽  
Chu Kong Liew ◽  
...  
Keyword(s):  
G Protein ◽  
Cultured Cells ◽  
Amino Terminus ◽  
Wild Type ◽  
Guanine Nucleotide Binding Protein ◽  
Inactive Form ◽  
In The Wild ◽  
Reporter Assays ◽  
Guanine Nucleotide Binding ◽  
G Protein Coupled

Little is known about the pharmacology or physiology of GPR37L1, a G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptor that is abundant in the cerebellum. Mice deficient in this receptor exhibit precocious cerebellar development and hypertension. We showed that GPR37L1 coupled to the G protein Gαs when heterologously expressed in cultured cells in the absence of any added ligand, whereas a mutant receptor that lacked the amino terminus was inactive. Conversely, inhibition of ADAMs (a disintegrin and metalloproteases) enhanced receptor activity, indicating that the presence of the amino terminus is necessary for GPR37L1 signaling. Metalloprotease-dependent processing of GPR37L1 was evident in rodent cerebellum, where we detected predominantly the cleaved, inactive form. However, comparison of the accumulation of cAMP (adenosine 3′,5′-monophosphate) in response to phosphodiesterase inhibition in cerebellar slice preparations from wild-type and GPR37L1-null mice showed that some constitutive signaling remained in the wild-type mice. In reporter assays of Gαs or Gαi signaling, the synthetic, prosaposin-derived peptide prosaptide (TX14A) did not increase GPR37L1 activity. Our data indicate that GPR37L1 may be a constitutively active receptor, or perhaps its ligand is present under the conditions that we used for analysis, and that the activity of this receptor is instead controlled by signals that regulate metalloprotease activity in the tissue.

Sign in / Sign up

Export Citation Format

Share Document