ISSN:1079-9893    

DOI:10.3109/10799899509045201

出版商:Taylor&Francis    

年代:1995

数据来源: Taylor

摘要:

AbstractReceptors that mediate their effects through G proteins are predicted to have a seven transmembrane domain architecture. The last few years have seen a remarkable increase in the cloning of members of this superfamily leading to the identification of many more receptors than previously thought to exist on the basis of differences in agonist and antagonist specificities. This has important implications for nomenclature and classification, especially in view of the difficulty in relating receptors identified through cloning techniques to endogenously expressed receptors. Receptor cloning has also identified important differences in receptors between species raising the question as to whether these should be considered as species homologues or distinct subtypes. It is also becoming increasingly apparent that the pharmacology of this superfamily of receptors is influenced by the nature of the G protein present in the host cell and by alternative splicing of the receptor. The rapid pace of developments in this area necessitate the need for a regular publication summarizing recent developments. In the future, the cloning of G protein-coupled receptors will enable rationalization of the naming of individual receptor subtypes and identification of their inter-relationships.

ISSN:1079-9893    

DOI:10.3109/10799899509045202

出版商:Taylor&Francis    

年代:1995

数据来源: Taylor

ISSN:1079-9893    

DOI:10.3109/10799899509045203

出版商:Taylor&Francis    

年代:1995

数据来源: Taylor

摘要:

AbstractThe expression of two G protein-coupled receptors was studied in several cell lines using the Semliki Forest virus expression system. Human neurokinin-2 and dopamine D3 receptor cDNAs were introduced into the pSFV1 vector.In vitrotranscribed RNAs were coelectroporated with pSFV-Helper RNA into BHK cells resulting inin vivopackaging of high titer SFV-NK-2 and SFV-D3 virus stocks, respectively. Infection of BHK, HOS and CHO cells with the recombinant NK-2 virus resulted in high levels of receptor expression as detected by metabolic labelling with [35S]-methionine. The expression of the NK-2 receptors in the cell membrane was demonstrated by Flow cytometry experiments on infected BHK and CHO cells with fluoresceinyl-NKA as the ligand. Saturation binding assays on membranes prepared from SFV-NK-2 infected CHO cells with [3H] GR100679 showed maximum receptor densities of 6.5 pmol receptor/ mg protein. Additionally, the expressed NK-2 receptors were able to stimulate Ca2+mobilization in CHO cells indicating functional coupling to G proteins. CHO cells infected with SFV-D3 also produced high levels of receptor as evidenced by both [35S]methionine labelling and [3H]spiperone binding.

ISSN:1079-9893    

DOI:10.3109/10799899509045204

出版商:Taylor&Francis    

年代:1995

数据来源: Taylor

摘要:

AbstractWe have produced a stable insect cell line derived fromSpodoptera frugiperda(Sf9) cells expressing a cDNA encoding aβ-subunit of theLymnaea stagnalisGABAAreceptor. The cDNA was randomly integrated into the insect cell genome under the control of a baculovirus immediate early gene (IE-1) promoter. Stable cell lines were established by transformation of Sf9 cells with the expression vector pIEK1.LGβ1 together with a plasmid encoding a selectable marker which confers neomycin (G418) resistance. Following growth in the presence of G418, neomycin resistant clones were selected, amplified and analysed for the presence of functional GABA-gated chloride channels. Electrophysiological analysis of one cell line showed the presence of a picrotoxin-sensitive chloride channel not present in control Sf9 cells. These channels were also sensitive to GABA, albeit at relatively high (mM) concentrations.

ISSN:1079-9893    

DOI:10.3109/10799899509045205

出版商:Taylor&Francis    

年代:1995

数据来源: Taylor

摘要:

AbstractStable expression of the MSH receptor in a homologous system is important for the study of the function and mechanism of signalling of this receptor. This is the first report on the stable expression of the humanα-MSH receptor in the mouse melanoma G4F clone which lacks an endogenous MSH receptor. Several stable transfectant cell lines were obtained all of which express the human MSH receptor in high numbers. Human MSH receptor mRNA expression was detected by Northern blot analysis. Competition binding experiments showed that the MSH receptors expressed in these cells have the same affinity for [Nle4,D-Phe7]-α-MSH as the MSH receptors of the human HBL melanoma cell line. Several of the transfectant cell lines produced melanin constitutively, some of them secreting melanin into the medium whereas other clones did not secrete melanin. MSH and cholera toxin did not or only marginally increase melanogenesis in these clones, and forskolin had an opposite effect. These results suggest that the human MSH receptor may be constitutively active in these transfected mouse melanoma cells.

ISSN:1079-9893    

DOI:10.3109/10799899509045206

出版商:Taylor&Francis    

年代:1995

数据来源: Taylor

摘要:

AbstractThe gene for the human m2 muscarinic receptor was expressed in Sf9 cells using the baculovirus expression system. As assessed by [3H]NMS binding, Sf9 cells expressed receptor at levels of 3.3 pmoles/mg protein. The receptor was identified on western blots using an anti-muscarinic receptor antibody and was shown to have the pharmacological characteristics of an m2 muscarinic receptor. Membranes from Sf9 cells were examined to identify endogenous G-proteins by immuno-blotting and by ADP-ribosylation, indicating the presence of Gq, and a pertussis-toxin substrate which was not recognised by antibodies raised against theα-subunits of Gi1, Gi2, Gi3 or Go. Gsαwas not detected, neither were there any cholera toxin substrates in Sf9 membranes. Sf9 membranes expressing m2 receptors did not show carbachol-stimulated GTPγS binding to endogenous G-proteins; however, when membranes were reconstituted with a mixture of purified Gi and Go, a maximum 8-fold stimulation of GTPγS binding was observed in response to carbachol that could be reduced by atropine. These data show that the human muscarinic m2 receptor expressed in Sf9 cells is functional.

ISSN:1079-9893    

DOI:10.3109/10799899509045207

出版商:Taylor&Francis    

年代:1995

数据来源: Taylor

摘要:

AbstractA plasmid vector has been constructed by insertion of the cDNA encoding theα1 subunit of the bovine GABAAreceptor into the LCR/MEL expression vector pNV1 downstream of the human globin locus control region between the promoter and the second intron of theβ-globin gene to produce pNVGABAα. This plasmid was transfected into murine erythroleukernia (MEL) cells using electroporation to obtain recombinant cells. Parental and recombinant cells were tested by both RNA dot blot and electrophysiological analysis for the the presence of bovine GABAAreceptorα1 subunit mRNA. Parental MEL cells did not express GABA-gated chloride channels but recombinant cells were sensitive to pressure-applied GABA. The GABA responses reversed at the equilibrium potential predicted for chloride ions. These results show that the al subunit of the bovine GAE3AA receptor inserts in the plasma membrane of the MEL cells and forms homo-oligomeric chloride channels that are gated by GABA. Our studies suggest, therefore, that the LCR/MEL system can be used for the expression of neurotransmitter receptor genes.

ISSN:1079-9893    

DOI:10.3109/10799899509045208

出版商:Taylor&Francis    

年代:1995

数据来源: Taylor

摘要:

AbstractReceptor phosphorylation is a key step in the process of rapid desensitization.β-adrenergic receptor kinase (βARK) is a specific receptor kinase that is known to phosphorylate and induce desensitization of several G-coupled receptors only when they are occupied by their agonists. In the present study we have done several modifications to the amino-terminal ofβARK1, in order to clarify its functional role. The recombinant mutants were tested for their ability to phosphorylate rhodopsin present in purified bovine ROS membranes which serves as a substrate forβARK1. Their expression levels were detected by Western blot analysis. We found that when the amino-terminal ofβARK1 is modified its expression level is very low, hence it is not able to phosphorylate over the basal. These findings suggest that this region is crucial for the normal processing of the protein.

ISSN:1079-9893    

DOI:10.3109/10799899509045209

出版商:Taylor&Francis    

年代:1995

数据来源: Taylor

摘要:

AbstractA cDNA clone for the human histamine H1receptor was isolated from a lung cDNA library and stably expressed in CHO cells. The recombinant receptor protein present in the cell membranes, displayed the functional and binding characteristics of histamine H1receptors. Mutation of Ser155 to Ala in the fourth transmembrane domain did not significantly change the affinity of the receptor for histamine and H1antagonists. However, mutation of the fifth transmembrane Asn198 to Ala resulted in a dramatic decrease of the affinity for histamine binding, and for the histamine-induced polyphosphoinositides breakdown, whereas the affinity towards antagonists was not significantly modified. In addition, mutation of another fifth transmembrane amino acid, Thr194 to Ala also diminished, but to a lesser extent, the affinity for histamine. These data led us to propose a molecular model for histamine interaction with the human H1receptor. In this model, the amide moiety of Asn198 and the hydroxyl group of Thr194 are involved in hydrogen bonding with the nitrogen atoms of the imidazole ring of histamine. Moreover, mutation of Thr194 to Ala demonstrated that this residue is responsible for the discrimination between enantiomers of cetirizine.

ISSN:1079-9893    

DOI:10.3109/10799899509045210

出版商:Taylor&Francis    

年代:1995

数据来源: Taylor