摘要:

AbstractBaby hamster kidney (BHK) cells transfected with an expression vector for the human thrombin receptor, and then treated with basic fibroblast growth factor, were found to express specific and saturable binding sites for biotinylated thrombin receptor peptide (SFLLRNPNDKYEPF). Analysis of the binding to live BHK cells yielded an equilibrium dissociation constant (Kd) of 3.0±0.3μmol/l and a maximal binding capacity (Bmax) of 31.0±0.5 nmol/mg of protein. In competitive binding experiments, the thrombin receptor agonist peptide (SFLLRN), which is a strong inducer of human platelet aggregation, was the most potent competitor. In contrast, position 1 to 2 inverted peptides such as FSLLRNPNDKYEPF and FSLLRNP, which fail to induce for the platelet aggregation, were less potent. This simple and convenient binding assay system using the biotinylated thrombin receptor peptide as a labeled ligand and the cloned thrombin receptor overexpressed in BHK cells may be useful for exploring specific antagonists of the receptor.

ISSN:1079-9893    

DOI:10.3109/10799899509045211

出版商:Taylor&Francis    

年代:1995

数据来源: Taylor

摘要:

AbstractThe rat vascular smooth muscle cell (VSMC) line A10 (ATCC CRL 1476) was stably transfected with a humanc-fospromoter-driven luciferase reporter gene to monitor thrombin receptor activation and subsequent induction ofc-fosexpression. Selective activation of the endogeneous thrombin receptor by the thrombin receptor activating peptide (TRAP1-6), SFLLRN, is shown here to result in a significant transient increase of intracellular [Ca2+], dose-dependent induction ofc-fospromoter-mediated luciferase activity, and stimulation of DNA synthesis. These data demonstrate that A10 cells and reporter line derivatives thereof possess a functional thrombin receptor very similar or identical to that previously described. Results obtained with various signal transduction modulating or inhibiting agents support previous notions showing that thrombin receptor activation by SFLLRN is coupled to events involving p21rasactivation, protein tyrosine kinase, and activation of PKC. The A10 reporter line described here proved to be a helpful and reliable tool to studyα-thrombin and TRAP1-6-mediated intracellular events, since it retained most of the spectrum of biological responses found in primary VSMC cultures.

ISSN:1079-9893    

DOI:10.3109/10799899509045212

出版商:Taylor&Francis    

年代:1995

数据来源: Taylor

摘要:

AbstractCurrently known disease-causing mutations in G protein coupled receptors are reviewed and discussed in conjunction with other naturally occurring receptor mutations. Special emphasis is made on opsin, vasopressin and MSH receptor mutations and what they tell are begining to tell us about the inner workings of this superfamily of signalling molecules.

ISSN:1079-9893    

DOI:10.3109/10799899509045213

出版商:Taylor&Francis    

年代:1995

数据来源: Taylor

ISSN:1079-9893    

DOI:10.3109/10799899509045214

出版商:Taylor&Francis    

年代:1995

数据来源: Taylor

摘要:

AbstractThe pharmacology of native and recombinant GABA-A receptors containing eitherγ1,γ2 orγ3 subunits has been investigated. The pharmacology of native receptors has been investigated by immunoprecipitating receptors from solubilised preparations of rat brain with antisera specific for individualγ-subunits and analysing their radioligand binding characteristics. Receptors containing aγ1-subunit do not bind benzodiazepine radioligands with high affinity. Those containing either aγ2 orγ3 subunit bind [3H]flumazenil with high affinity. Some compounds compete for these binding sites with multiple affinities, reflecting the presence of populations of receptors containing several different types ofα-subunit. Photoaffinity-labelling of GABA-A receptors from a cell line stably expressing GABA-A receptors of compositionα1β3γ2 followed by immunoprecipitation of individual subunits revealed that theαandγbut not theβ-subunit could be irreversibly labelled by [3H]flunitrazepam.The properties of recombinant receptors have been investigated in oocytes expressingγ1,γ2, orγ3 subunits in combination with anαand aβ-subunit. Some compounds such as zolpidem, DMCM and flunitrazepam show selectivity for receptors containing differentγ-subunits. Others such as CL 218,872 show no selectivity between receptors containing differentγ-subunits but exhibit selectivity for receptors containing differentα-subunits. These data taken together suggest that the benzodiazepine site of the GABA-A receptor is formed with contributions from both theαandγ-subunits.

ISSN:1079-9893    

DOI:10.3109/10799899509045215

出版商:Taylor&Francis    

年代:1995

数据来源: Taylor

摘要:

AbstractIn order to map in detail the ligand binding sites of fibroblast growth factor receptor 2 (FGFR2) and keratinocyte growth factor receptor (KGFR), we have generated receptor molecules that are chimeric within the membrane proximal sequence that varies between them. The chimeric molecules are found to bind aFGF with a greater than 5-fold difference in affinity, indicating that there is coupling between the chimeric regions with respect to aFGF binding. Further, binding of bFGF and KGF is abolished in the chimeras, showing that the binding site for these ligands requires the whole of the 48- or 50- amino acid variable sequence to be intact. Direct interactions between the different regions exchanged in the chimeras are most probably involved in forming KGF or bFGF binding sites.

ISSN:1079-9893    

DOI:10.3109/10799899509045216

出版商:Taylor&Francis    

年代:1995

数据来源: Taylor

摘要:

Abstract[35S]-GTPγS binding has been used to study the function of cloned human 5-HT1Dreceptor subtypes stably expressed in chinese hamster ovary (CHO) cells. 5-HT stimulated [35S]-GTPγS binding to membranes from cells expressing 5-HT1Dαor 5-HT1Dβreceptors. In membranes containing 5-HT1Dβreceptors, 5-CT and sumatriptan stimulated binding to a similar extent as 5-HT while yohimbine, metergoline and 8-OHDPAT were partial agonists. The order of potency for agonists was 5-CT>5-HT>metergoline>sumatriptan>yohimbine>8-OHDPAT. The stimulation of binding by 5-HT in membranes containing 5-HT1Dβreceptors was potently antagonised by methiothepin (pA28.9±0.1). The overall pharmacological profile for the human 5-HT1Dβreceptor, defined using [35S]-GTPγS binding, agreed well with that reported for inhibition of forskolin-stimulated adenylyl cyclase. In addition, methiothepin and ketanserin inhibited basal [35S]-GTPγS binding to membranes containing 5-HT1Dαor 5-HT1Dβreceptors, suggesting that these compounds show negative efficacy at 5-HT1Dreceptor subtypes. The data show that [35S]-GTPγS binding is a suitable method for studying the interaction between cloned human 5-HT1Dreceptors and G-proteins.

ISSN:1079-9893    

DOI:10.3109/10799899509045217

出版商:Taylor&Francis    

年代:1995

数据来源: Taylor

摘要:

AbstractFindings from the last two years in signal transduction research, including the elucidation of the crystal structure ofα1, the uncovering of multiple roles for lipidation, the mimicry of receptor action with peptides, and both thein vitroreconstitution of inhibition of adenylyl cyclase and thein cellreconstitution of receptor-G protein coupling in transient and stable expression studies, are integrated into a“current”view of the receptor→G protein→effector pathway. The question is raised whether receptor orβγis the nucleotide exchange factor, and the central participation of Mg2+in G protein activation and the change in affinity of the G protein for Mg2+during receptor-stimulated activation are stressed.

ISSN:1079-9893    

DOI:10.3109/10799899509045218

出版商:Taylor&Francis    

年代:1995

数据来源: Taylor

摘要:

AbstractIt is now appreciated both that G-protein-linked receptors and signal transducing heterotrimeric G-proteins consist of large multi-member superfamilies and that regulation of a signal transduction cascade can be produced by a variety of means following activation of a G-protein by a receptor. To begin to unravel the complexities of this regulation it is clearly important to be able to define the molecular identity of the G-protein or G-proteins activated by a receptor and to assess the quantitative importance of such interactions for the integration of signals produced by a receptor agonist. Substantial progress has been made towards these goals in recent years and the purpose of this short review will be to discuss the use and potential limitations of some of the currently most widely used approaches.

ISSN:1079-9893    

DOI:10.3109/10799899509045219

出版商:Taylor&Francis    

年代:1995

数据来源: Taylor

摘要:

AbstractThe D2dopamine receptor is known to be functionally coupled to the inhibition of adenylate cyclase when expressed in a number of mammalian cell lines. However, functional coupling of the recently discovered D3and D4dopamine receptor subtypes has been more difficult to demonstrate. In this study, human D2, D3and D4receptors were stably expressed separately in human embryonic kidney cells (HEK 293). In these cells, activation of D2, D3or D4receptors resulted in the inhibition of forskolin-stirnulated adenylate cyclase activity in a dose responsive manner. This activation was prevented by pre-incubation of the cells expressing these receptors with the dopaminergic antagonist haloperidol. Radioligand binding studies using [3H]spiperone confirmed that the atypical neuroleptic clozapine has higher affinity for the human D4receptor than the D3or D4receptors, although only 6-fold higher than the D2receptor in this study. In addition, ribonuclease protection studies demonstrated the presence of D4dopamine receptor mRNA in human brain regions.

ISSN:1079-9893    

DOI:10.3109/10799899509045220

出版商:Taylor&Francis    

年代:1995

数据来源: Taylor