BiochemicalSocietyTransactions(1 992)20 147s Receptor-apecific antisera as probes for tho D,-dopuine receptor. A.J. DOHERTY and P.G. STRANGE, Biological Laboratory, The Canterbury, Kent, CT2 7NJ, UK.
University,
D,-dopamine receptors ( D2DR) belong to the superfamily of G-protein coupled receptors and are thought to contain seven membrane spanning a-helices. Molecular cloning has revealed two splice variants of this receptor [ 11 which differ by a 29-amino acid sequence inserted into the third intracellular loop but which at present appear to have identical pharmacological profiles. These two receptor sub-types have been designated D2(-) (without the insert) (with the insert). Thus there is a and D2-, need for receptor sub-type specific probes with which to map the relative distribution of the D,-, and D2(-) receptors. Polyclonal antibodies raised against peptides from regions of the receptor(s) with little or no homology could be invaluable tools as such probes. Furthermore, such antibodies can throw light on the membrane organisation of receptors such as the D,DR and on regions of the protein structure which are important for the functions of such receptors. This laboratory has previously reported the preparation of an antiserum raised against a peptide (AAFtRAQELEMEML) in the third intracellular loop of the D,DR [2] which recognises a 95kDa species on western blots using a horse radish peroxidase conjugated second antibody in bovine caudate nucleus, a brain region rich in D2DR, but not in cerebellum, in which little or no D,DR is found. Furthermore, this antiserum (AS1) recognises both native and denatured D,DR [2] and also interrupts G-protein coupling to solubilised receptor preparations [3]. We now report the initial characterisation of two further antisera raised against peptides from the second extracellular loop (AS3) and insert (AS4) of the two D,DR receptor sub-types. Antiserum AS3 should therefore recognise both ,,,,D and D,-, while AS4 should recognise only D,-,, Antisera were raised in New Zealand White rabbits by injection of synthetic peptides conjugated to keyhole limpet haemocyanin and either complete (first injection) or incomplete (subsequent injections) Freund's adjuvant. Antiserum AS3 was raised against the peptide NNTDQNECIIAN while AS4 was raised against the peptide GNCTHPEDMKLCTV. The specificity of the antisera was determined by western blotting of bovine caudate nucleus and cerebellum membranes using an alkaline phosphatase conjugated goat anti-rabbit IgG as second anti body Using this system, AS3 was shown to label three protein bands of interest. TWO, centred at llOkDa and 80kDa, were found only in caudate nucleus while a band centred at 95kDa was seen in both caudate nucleus and cerebellum. The intensity of this last band in the cerebellum was much less than that in the caudate nucleus and was also present when pre-immune serum was used. Similar
.
results have been found with AS1. Furthermore, parallel experiments using AS1 and AS3 with the horse radish peroxidase and alkaline phosphatase conjugated second antibodies showed that the 95kDa bands seen with the two systems were the same. Thus this band presumably represents the major D,DR sub-type present in bovine brain. Similar experiments have been performed with AS4. Using this antiserum, caudate nucleus specific proteins have been detected centred at lO5kDa and 77kDa but no bands have been located at 95kDa. All of the above antisera also label a high molecular weight protein (Sl6OkDa) specific to the caudate nucleus. These results suggest that the 95kDa species labelled by AS1 and AS3 represents D,,, while D200s, is represented by the bands at either 105-110kDa or 77-80kDa. Support for the former comes from studies with CHO cells transfected with the D, cDNA (CH06, S.Castro & P.G.Strange, unp3lished). In this case, AS3 and AS4 label a protein of 102-103kDa in the transfected cells but not the untransfected cells. Work is now in progress to determine the specificity of these antisera in LTK- cells transfected with D,(,, [4] and to determine the effect of antisera 1,3 and 4 on the coupling of Gprotein to the D,DR transfected into these two cell lines. Initial studies have also been performed to determine the usefulness of these antisera for the detection of D,DR in situ using CH06 cells in indirect immunofluorescence experiments. Cells were fixed in methanol at -2OOC and incubated with AS1 overnight at 4OC followed by incubation with goat anti-rabbit IgG conjugated with fluorescein isothiocyanate and viewed under W light. Transfected cells could be labelled with a much greater intensity than untransfected cells demonstrating that this antiserum can label native receptor within a membrane environment. Work is now in progress to use all the antisera in similar experiments to determine the membrane topography of the D,DR in CH06 cells. The results presented above suggest that the antisera raised in our laboratory are able to distinguish between D,-, and D,(,,,,, in brain membranes and that it may now be possible to provide direct experimental evidence for the topography and membrane organisation of D,DR when present in a membrane environment. We thank the Medical Research Council for financial support. 1. Giros b., Sokoloff P., Martes M-P., Riou J-F., Emorine L.J. & Schwartz J-C. (1989) Nature 3 4 2 # 923-926 2. Chazot P.L., Wilkins M. & Strange P.G. (1991) Biochem. SOC. Trans. 19# 143s 3. Strange P.G. (1991) Biochem. SOC. Trans. in press 4. Bunzow J.R., Van To1 H.H.M. , Grandy D.K. , Albert P., Salon J., Christie M., Machida C.A., Neve K.A. & Civelli 0. (1988) Nature 336# 783-787
University,
D,-dopamine receptors ( D2DR) belong to the superfamily of G-protein coupled receptors and are thought to contain seven membrane spanning a-helices. Molecular cloning has revealed two splice variants of this receptor [ 11 which differ by a 29-amino acid sequence inserted into the third intracellular loop but which at present appear to have identical pharmacological profiles. These two receptor sub-types have been designated D2(-) (without the insert) (with the insert). Thus there is a and D2-, need for receptor sub-type specific probes with which to map the relative distribution of the D,-, and D2(-) receptors. Polyclonal antibodies raised against peptides from regions of the receptor(s) with little or no homology could be invaluable tools as such probes. Furthermore, such antibodies can throw light on the membrane organisation of receptors such as the D,DR and on regions of the protein structure which are important for the functions of such receptors. This laboratory has previously reported the preparation of an antiserum raised against a peptide (AAFtRAQELEMEML) in the third intracellular loop of the D,DR [2] which recognises a 95kDa species on western blots using a horse radish peroxidase conjugated second antibody in bovine caudate nucleus, a brain region rich in D2DR, but not in cerebellum, in which little or no D,DR is found. Furthermore, this antiserum (AS1) recognises both native and denatured D,DR [2] and also interrupts G-protein coupling to solubilised receptor preparations [3]. We now report the initial characterisation of two further antisera raised against peptides from the second extracellular loop (AS3) and insert (AS4) of the two D,DR receptor sub-types. Antiserum AS3 should therefore recognise both ,,,,D and D,-, while AS4 should recognise only D,-,, Antisera were raised in New Zealand White rabbits by injection of synthetic peptides conjugated to keyhole limpet haemocyanin and either complete (first injection) or incomplete (subsequent injections) Freund's adjuvant. Antiserum AS3 was raised against the peptide NNTDQNECIIAN while AS4 was raised against the peptide GNCTHPEDMKLCTV. The specificity of the antisera was determined by western blotting of bovine caudate nucleus and cerebellum membranes using an alkaline phosphatase conjugated goat anti-rabbit IgG as second anti body Using this system, AS3 was shown to label three protein bands of interest. TWO, centred at llOkDa and 80kDa, were found only in caudate nucleus while a band centred at 95kDa was seen in both caudate nucleus and cerebellum. The intensity of this last band in the cerebellum was much less than that in the caudate nucleus and was also present when pre-immune serum was used. Similar
.
results have been found with AS1. Furthermore, parallel experiments using AS1 and AS3 with the horse radish peroxidase and alkaline phosphatase conjugated second antibodies showed that the 95kDa bands seen with the two systems were the same. Thus this band presumably represents the major D,DR sub-type present in bovine brain. Similar experiments have been performed with AS4. Using this antiserum, caudate nucleus specific proteins have been detected centred at lO5kDa and 77kDa but no bands have been located at 95kDa. All of the above antisera also label a high molecular weight protein (Sl6OkDa) specific to the caudate nucleus. These results suggest that the 95kDa species labelled by AS1 and AS3 represents D,,, while D200s, is represented by the bands at either 105-110kDa or 77-80kDa. Support for the former comes from studies with CHO cells transfected with the D, cDNA (CH06, S.Castro & P.G.Strange, unp3lished). In this case, AS3 and AS4 label a protein of 102-103kDa in the transfected cells but not the untransfected cells. Work is now in progress to determine the specificity of these antisera in LTK- cells transfected with D,(,, [4] and to determine the effect of antisera 1,3 and 4 on the coupling of Gprotein to the D,DR transfected into these two cell lines. Initial studies have also been performed to determine the usefulness of these antisera for the detection of D,DR in situ using CH06 cells in indirect immunofluorescence experiments. Cells were fixed in methanol at -2OOC and incubated with AS1 overnight at 4OC followed by incubation with goat anti-rabbit IgG conjugated with fluorescein isothiocyanate and viewed under W light. Transfected cells could be labelled with a much greater intensity than untransfected cells demonstrating that this antiserum can label native receptor within a membrane environment. Work is now in progress to use all the antisera in similar experiments to determine the membrane topography of the D,DR in CH06 cells. The results presented above suggest that the antisera raised in our laboratory are able to distinguish between D,-, and D,(,,,,, in brain membranes and that it may now be possible to provide direct experimental evidence for the topography and membrane organisation of D,DR when present in a membrane environment. We thank the Medical Research Council for financial support. 1. Giros b., Sokoloff P., Martes M-P., Riou J-F., Emorine L.J. & Schwartz J-C. (1989) Nature 3 4 2 # 923-926 2. Chazot P.L., Wilkins M. & Strange P.G. (1991) Biochem. SOC. Trans. 19# 143s 3. Strange P.G. (1991) Biochem. SOC. Trans. in press 4. Bunzow J.R., Van To1 H.H.M. , Grandy D.K. , Albert P., Salon J., Christie M., Machida C.A., Neve K.A. & Civelli 0. (1988) Nature 336# 783-787
