Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Guanine nucleotide-binding protein-coupled receptors have been shown to exist in both a high affinity agonist (HiAg) and a low affinity agonist (LowAg) state. The formation of the HiAg state is promoted by agonists, and the formation of this state of the receptor appears to be a critical factor in the generation of the effector-activating complex G alpha.GTP.Mg2+ and in the production of a stimulus. The magnitude of the difference in the affinity a compound has for the HiAg versus the LowAg state of the receptor has been related to the intrinsic activity of the compound. In this paper the HiAg and LowAg affinities (Ki) of full and partial dopamine agonists of varying levels of intrinsic activity were determined using membranes from Chinese hamster ovary cells stably transfected with the D2i receptor. The HiAg state was defined using the recently described dopamine agonist ligand [3H]U-86170, and the LowAg state was defined using [3H] raclopride plus 600 microM GTP. The LowAg/HiAg ratios for apomorphine (43), HW-165 (12.5), (-)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine [(-)-3-PPP] (4.5), terguride (1.6), SDZ-208-911 (1.2), and SDZ-208-912 (0.3) were found to correlate well with their electrophysiologically derived intrinsic activities (r = 0.92). Using this relationship, the intrinsic activity for compounds such as (+)-3-PPP (112%), quinpirole (104%), U-68553B (102%), and U-86170 (95%) was predicted to be high (greater than 90%); (-)-apomorphine (73%) was of high/moderate intrinsic activity, HW-165 (52%), (+)-apomorphine (51%), and (-)-3-PPP (34%) were in the intermediate range, and terguride (16.5%), SDZ-208-911 (11.7%), and SDZ-208-912 (-12%) were at the lower end of the intrinsic activity spectrum. The receptor state binding-determined intrinsic activity values for quinpirole (100%), U-86170F (94.8%), HW-165 (52.1%), (-)-3-PPP (34.3%), SDZ-208-911 (11.7%), and SDZ-208-912 (-12%) were found to correlate well (r = 0.908) with their maximum response (intrinsic activity), as determined using ATP-mediated increases in arachidonic acid release from CHO-D2i cells. In addition, the maximal effect of several of these compounds on rat striatal homovanillic acid (HVA) levels was determined. The drug-induced changes in tissue HVA levels were found to be consistent with the affinity-derived intrinsic activities of the drugs.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Pharmacol 1992 Sep
PMID:Intrinsic activity determinations at the dopamine D2 guanine nucleotide-binding protein-coupled receptor: utilization of receptor state binding affinities. 135 42

Small GTP-binding proteins are encoded by ras-like genes and play a central role in cell differentiation and membrane vesicle transport. By screening genomic and cDNA libraries of the Ascomycete fungus Neurospora crassa with Zmypt genes from Zea mays we have isolated a member of the ypt gene family, Ncypt1. The gene resides on a 4 kb fragment of genomic DNA and contains four introns, which interrupt the coding sequence of a protein of 203 amino acid residues. The Ncytp1 gene was assigned to a single-copy gene encoding a transcript of 1.5 kb and a protein of 26,000 daltons. The gene maps on linkage group IIR between DB0001 and ccg-2 close to the Fsr-3 locus. Analysis of the nucleotide sequence and the deduced protein sequence revealed a striking homology to yeast, mouse and human genes encoding small GTP-binding proteins that are related to the ras supergene family. Homology was most significant to ypt1 from Schizosaccharomyces pombe, Mus musculus and Homo sapiens sharing 84.8%, 82.3%, and 82.3% identity, respectively. Common domains present in other small GTP-binding proteins were identified in the predicted sequence of the NCYPT1 protein, and the arrangement of peptide motifs sharing similarity with well characterized, small GTP-binding proteins suggests that the NCYPT1 protein is a GTPase. The C-terminal region extending from amino acid residues 175 to 199 shares only weak amino acid sequence similarity with other eukaryotic GTPases. Like other RAS proteins the NCYPT1 protein contains two conserved C-terminal cysteine residues, suggesting post-translational modification(s) by fatty acylation required for membrane anchoring. The high degree of homology between the NCYPT1 protein and eukaryotic YPT proteins suggests that NCYPT1 could be involved in the control of secretory processes.
Mol Gen Genet 1992 Nov
PMID:The Ncypt1 gene from Neurospora crassa is located on chromosome 2: molecular cloning and structural analysis. 136 Dec 12

Using the polymerase chain reaction technique with degenerative primers, we obtained from a rat pituitary cDNA library a cDNA fragment, rAP236, that exhibited considerable homology to known receptors that belong to the guanine nucleotide-binding protein (G protein)-coupled receptor superfamily. Oligonucleotides to this fragment were used as probes to obtain a full-length cDNA from the rat pituitary cDNA library. This clone, rAP6-26, encoded a 383-amino acid protein with seven putative transmembrane domains that are characteristic of G protein-coupled receptors. The predicted amino acid sequence of the rAP6-26 cDNA exhibits 56-66% homology to recently cloned somatostatin (SRIF) receptors. Membranes prepared from COS-7 cells transfected with the rAP6-26 cDNA showed specific binding of 125I-Tyr11-SRIF, thus identifying the cDNA clone as a novel SRIF receptor. Radioligand binding competition analysis using somatostatin-28 (SRIF-28) and a number of cyclic SRIF analogs revealed that SRIF-28 was the most potent competitor of 125I-Tyr11-SRIF binding, with a approximately 30-fold greater affinity for the receptor than that of SRIF. In addition, binding of 125I-Tyr11-SRIF was markedly reduced in the presence of Na+ ions and GTP, indicating coupling of rAP6-26 receptors to inhibitory G proteins in COS-7 membranes. In adenylyl cyclase assays, forskolin-induced cAMP accumulation was inhibited by SRIF and SRIF-28, thus confirming that the rAP6-26 cDNA encodes a functional receptor protein. By Northern blot analysis, a approximately 2.6 kilobase mRNA encoding the receptor was present in the pituitary but not in the liver, small intestine, kidney, pancreas, cerebellum, or cortex. Lack of receptor mRNA expression in the brain was confirmed by in situ hybridization histochemical studies. Thus, we report the cloning of a novel rat pituitary SRIF receptor, termed SSTR4, that has marked preferential affinity for SRIF-28 and is linked to inhibition of adenylyl cyclase.
Mol Pharmacol 1992 Dec
PMID:Molecular cloning and expression of a pituitary somatostatin receptor with preferential affinity for somatostatin-28. 826 65

Two distinct GAPs of 120 and 235 kDa called GAP1 and NF1 serve as attenuators of Ras, a member of GTP-dependent signal transducers, by stimulating its intrinsic guanosine triphosphatase (GTPase) activity. The GAP1 (also called Ras GAP) is highly specific for Ras and does not stimulate the intrinsic GTPase activity of Rap1 or Rho. Using GAP1C, the C-terminal GTPase activating domain (residues 720-1044) of bovine GAP1, we have shown previously that the GAP1 specificity is determined by the Ras domain (residues 61-65) where Gln61 plays the primary role. The corresponding domain (residues 1175-1531) of human NF1 (called NF1C), which shares only 26% sequence identity with the GAP1C, also activates Ras GTPases. In this article, we demonstrate that the NF1C, like the GAP1C, is highly specific for Ras and does not activate either Rap1 or Rho GTPases. Furthermore, using a series of chimeric Ras/Rap1 and mutated Ras GTPases, we show that Gln at position 61 of the GTPases primarily determines that NF1C as well as GAP1C activates Ras GTPases, but not Rap1 GTPases, and Glu at position 63 of the GTPases is required for maximizing the sensitivity of Ras GTPases to both NF1C and GAP1C. Interestingly, replacement of Glu63 of c-HaRas by Lys reduces its intrinsic GTPase activity and abolishes the GTPase activation by both NF1C and GAP1C. Thus, the potentiation of oncogenicity by Lys63 mutation of c-HaRas appears primarily to be due to the loss of its sensitivity to the two major Ras signal attenuators (NF1 and GAP1).
Mol Biol Cell 1992 Dec
PMID:The role of Gln61 and Glu63 of Ras GTPases in their activation by NF1 and Ras GAP. 136 1

The mechanism by which intracellularly applied guanosine-5'-O-(2-thiodiphosphate) alters responses to chicken II luteinizing hormone-releasing hormone, muscarine, and substance P in bullfrog sympathetic neurons was examined. Whole-cell recordings were made from enzymatically dissociated single neurons. Guanosine-5'-O-(2-thiodiphosphate) was applied intracellularly by adding it to the pipette solution with fixed amounts of GTP. Guanosine-5'-O-(2-thiodiphosphate) did not affect the proportion of cells that responded to any of the agonists. Guanosine-5'-O-(2-thiodiphosphate) decreased the amplitude of the responses to submaximal concentrations of agonist. At maximal concentrations of agonist, guanosine-5'-O-(2-thiodiphosphate) did not decrease the response to the first application of agonist; however, with guanosine-5'-O-(2-thiodiphosphate) intracellularly, successive responses to maximal concentrations of agonist were decreased in amplitude and increased in time course. Intracellular guanosine-5'-O-(2-thiodiphosphate) did not accelerate the rate or magnitude of desensitization to substance P. A kinetic model of receptor-guanine nucleotide-binding protein (G protein) coupling predicts that a decrease in the available G protein pool should decrease both the magnitude and the time course of the build-up of active G proteins. The results are consistent with the hypothesis that guanosine-5'-O-(2-thiodiphosphate) binds tightly to G proteins, thereby effectively decreasing the available G protein pool with repeated agonist applications.
Mol Pharmacol 1992 Mar
PMID:Intracellular guanosine-5'-O-(2-thiodiphosphate) alters the dynamics of receptor-mediated responses in bullfrog sympathetic neurons. 137 87

We have shown that the second intron of the Podospora mitochondrial gene coding for cytochrome b (Cytb 12) splices autocatalytically, using in vitro transcripts generated from the T7 promoter. The reaction takes place at 37 degrees C in the presence of 50 mM TRIS-HCl pH 7.5, 60 mM MgCl2 and 1 mM GTP but shows a low efficiency even at high KCl concentrations of up to 1.2 M. Under these conditions, intron bI2 follows the conventional pathway of group I splicing, and all characteristic products, with regard to both transesterification and hydrolysis, could be identified. Moreover, the intron is capable of undergoing cyclization, thereby releasing the noncoded G and one additional nucleotide (U) from the 5' end. The 5' cleavage site is preceded by the same two nucleotides, indicating a base-pairing at the same site of the internal guide sequence (IGS) for both splicing and cyclization ("one-binding-site model"). In addition, products resulting from site-specific hydrolysis 138 nucleotides downstream of the 5' splice site were detected. Unusually, the shortened intron is also able to form a circular RNA and an alternative sequence that aligns the cyclization site to the catalytic core of the intron must be assumed.
Mol Gen Genet 1992 May
PMID:Self-splicing of a mitochondrial group I intron from the cytochrome b gene of the ascomycete Podospora anserina. 137 8

We have synthesized a potent, selective, radioiodinated antagonist of the human neurokinin-1 (NK1) receptor and have characterized its binding to the cloned receptor expressed in Chinese hamster ovary cells. (cis)-2-(Diphenylmethyl)-N-[(2-iodophenyl)-methyl]-1- azabicyclo[2.2.2]octan-3-amine (L-703606) inhibits binding of 125I-Tyr8-substance P to the human NK1 receptor with an IC50 of 2 nM. This compound is a competitive antagonist of substance P-induced inositol phosphate generation, with a Kb of 29 nM. [125I]L-703606 binds to a single class of high affinity binding sites in human NK1/Chinese hamster ovary cell membranes (Kd = 0.3 nM). Substance P inhibits the binding of [125I]L-703606 to 65% of the NK1 receptor sites with a Kd of 0.04 +/- 0.03 nM and to the remaining 35% of the sites with a Kd of 1.5 +/- 0.7 nM. Addition of the nonhydrolyzable GTP analog guanylyl-5'-(beta, gamma-imido)diphosphate [Gpp(NH)p] shifts greater than 90% of the binding sites to the lower affinity state. In addition, Gpp(NH)p markedly alters the dissociation of substance P from the NK1 receptor by increasing the number of sites in the low affinity, rapidly dissociating state. However, Gpp(NH)p does not affect the rate of dissociation of [125I]L-703606. These data suggest that the pharmacological properties of [125I]L-703606 binding to the human NK1 receptor are similar to those of antagonists of nonpeptide guanine nucleotide-binding protein-coupled receptors and that this ligand will be useful for the biochemical and pharmacological characterization of the human NK1 receptor.
Mol Pharmacol 1992 Sep
PMID:Characterization of the binding of a potent, selective, radioiodinated antagonist to the human neurokinin-1 receptor. 138 85

This work explored the role of the cholinergic pathway, assessed at a post-synaptic level by the use of isolated smooth muscle cells, in the impairment of antral motility associated with diabetic gastroparesis. Contractile response to carbachol--but not to erythromycin, a motilin receptor agonist--was abolished in antral smooth muscle cells isolated from (i) rats previously rendered diabetic by a single i.v. dose of streptozotocin (STZ, 60 mg/kg) and (ii) db/db spontaneously diabetic mice. Insulin treatment of STZ-rats was able to prevent the impairment of the carbachol contractile response, but not to reverse it once established. In STZ-rats, impairment of contractile response was not associated with a change in density of [3H]-N-methyl-scopolamine ([3H]-NMS) binding sites (approximately 1.5 fmol/mg protein). Displacement curve of the [3H]-NMS binding by carbachol was shifted to the right in diabetic rats as compared to controls. The addition of GTP-gamma-S induced a shift to the right of the displacement curve in control but not in diabetic animals. These results strongly suggest that diabetes is associated with an early and specific alteration of the muscarinic control of contraction of antral smooth muscles at a post-synaptic level, associated with an alteration of the GTP-binding proteins coupled to muscarinic receptors.
Mol Cell Biochem 1992 Feb 12
PMID:Impairment of contractile response to carbachol and muscarinic receptor coupling in gastric antral smooth muscle cells isolated from diabetic streptozotocin-treated rats and db/db mice. 138 42

To investigate the modulation of tracheal ciliary beat frequency (CBFt) by purine nucleotides and nucleosides acting on luminal receptors, aerosolized ATP, GTP, AMP-PNP, GMP-PNP, adenosine, and guanosine were each administered separately to the tracheal lumen in eucapnically ventilated, barbiturate-anesthetized beagles. Four studies were conducted in each of seven dogs from a cohort of eight dogs. The CBFt responses were measured on the right lateral surface of the mid-trachea using heterodyne mode correlation analysis laser light scattering. Aerosolized 10(-6) M and 10(-5) M ATP stimulated CBFt from the baseline of 5.9 +/- 1.4 Hz to maxima of 12.1 +/- 1.4 Hz and 13.3 +/- 1.6 Hz, respectively, while the same corresponding ATP-analogue (AMP-PNP) concentrations stimulated baseline CBFt to maxima of 12.7 +/- 4.1 Hz and 18.1 +/- 2.1 Hz, respectively. Similarly, 10(-6) M and 10(-5) M GTP stimulated baseline CBFt to maxima of 14.8 +/- 1.1 Hz and 12.8 +/- 4.6 Hz, respectively. The corresponding GTP-analogue (GMP-PNP) concentrations stimulated CBFt to maxima of 14.5 +/- 2.1 Hz and 18.8 +/- 4.4 Hz, respectively. Prior delivery of 10(-5) M adenosine reduced all these nucleotide-induced stimulatory responses. Prior delivery of 10(-5) guanosine partially reduced the GTP- and the GMP-PNP-induced stimulatory responses. These data demonstrate that nucleotides and nucleosides modulate CBFt through specific P2 and P1 purinergic receptors on the luminal surface, thus providing a direct mechanism within the airways to enhance the transport of mucus.
Am J Respir Cell Mol Biol 1992 Oct
PMID:Luminal purinergic regulatory mechanisms of tracheal ciliary beat frequency. 138 12

1. A group of 21 to 24-kDa proteins of pheochromocytoma (PC-12) cells was found in blot overlay assays to bind specifically [alpha-32P]GTP. Binding was inhibited by GTP analogues but not by ATP. Such small GTP-binding proteins were found in the cytosolic and in the particulate fraction of the cells, but they were unevenly distributed: about 75% of the small GTP-binding proteins were localized within the particulate fraction of the cells. Separation of these proteins by two-dimensional gel electrophoresis revealed the existence of seven distinct [alpha-32P]GTP-binding proteins. 2. Targeting of the small GTP-binding proteins to the particulate fraction of PC-12 cells requires modification by isoprenoids, since depleting the cells of the isoprenoid precursor mevalonic acid (MVA) by the use of lovastatin resulted in a 50% decrease in membrane-bound small GTP-binding proteins, with a proportionate increase in the cytosolic form. This blocking effect of lovastatin was reversed by exogenously added MVA. 3. In addition, metabolic labeling of PC-12 cells with [3H]MVA revealed incorporation of [3H]MVA metabolites into the cluster of 21 to 24-kDa proteins in a form typical of isoprenoids; the label was not removed from the proteins by hydroxylamine, and labeling was enhanced in cells incubated with lovastatin. The latter effect reflects a decrease in the isotopic dilution of the exogenously added [3H]MVA, as the addition of exogenous MVA reversed the effect of lovastatin on [3H]MVA-metabolite incorporation into the 21 to 24-kDa proteins. 4. Additional experiments demonstrated that isoprenylation is required not only for membrane association of small GTP-binding proteins, but also for their further modification by a methylation enzyme. This was evident in experiments in which the cells were metabolically labeled with [methyl-3H]methionine, a methylation precursor. The group of 21 to 24-kDa proteins was labeled with a methyl-3H group in a form typical of C-terminal-cysteinyl carboxylmethyl esters. Their methylation was blocked by the methylation inhibitors methylthioadenosine (MTA), 3-deazadenosine and homocysteine thiolactone as well as by lovastatin. MVA reversed the lovastatin block of methylation. 5. Two-dimensional gel analysis of the [3H]methylated proteins detected seven methylated small GTP-binding proteins that correspond to the isoprenylated proteins. Levels of the small GTP-binding proteins as well as isoprenylation and methylation were reduced by cycloheximide. 6. Distribution of the methylated proteins between particulate and cytosolic fractions was found to be similar to that of the small GTP-binding proteins (i.e., a 4:1 ratio).(ABSTRACT TRUNCATED AT 400 WORDS)
Cell Mol Neurobiol 1992 Aug
PMID:Isoprenylation and carboxylmethylation in small GTP-binding proteins of pheochromocytoma (PC-12) cells. 139 71


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