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A novel Gs protein in the striatum
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A novd Gs prote'm in the stHatum Vincent, S.R., Drinnan, S.L. and Hope, B.T. K'msz~en Laboratoryof NeurologicalResearch, Department of Psychiatry, The University of British Columbi~ Vancouver, B.C., V6T 1WS, Canada
Many hormone and neurotransmitter receptors are coupled to adenylyl cyclase by the GTP-binding protein Gs. In the striatum, activation of D1 dopamine receptors results in a stimulation of adenylyl cyclase. Both the binding of D1 agonists and the stimulation of adenylyl cyclase are dependent upon GTP, implicating Gs in this action. However, the dopamine-stimulated adenylyl cyclase appears to have properties distinct from other agonist-stimulated adenylyl cyclases. Dopamine stimulation is readily observed in both brain homogena~es and slices, while fl-adrenergic stimulation, which can be detected in slices, is lost upon homogenization. This suggests that the D1 and fl-adrenergic ~to~ are coupled to adenylyl cyclase in distinct ways. We have used a synthetic oligonucleotide probe to the cholera toxin ADP-ribosylation site of the alpha subunit of Gs, to det~.t the mRNA for this protein in the rat brain. Northern blot analysis of whole rat brain mRNA indicated that a single mRNA was present of about 1900 bases. This is consistent with findings in other tissues. This message was not detectable in the striation. This probe was also used for the in situ localization of Gs-a mRNA in the rat brain. A regionally selective distribution was found, with high levels in the cortex, hippocampus and cerebellum. Of particular interest was the very low level of signal in the striatum. A closer examination of the distribution of Gs-a mRNA in the forebrain indicated that the regions in which this message was not detectable corresponded to those areas receiving a major dopamine innervation from the midbrain; the caudate-pmamen, nucleus accumbens and oifactory tubercle. Cholera toxin ADP-ribosylates the alpha subunit of Gs, thereby activating this protein. In striatal membranes, cholera toxin results in the ADP-ribosylation of a major band of 45 kD, and a minor band of 52 kD. This is opposite to the pattern of ADP-ribosylated proteins observed in other brain regions. These results indicate that a novel form of Gs-a is present in the striatum where it may act to couple the D1 receptor to adenylyl cyclase. Multiple forms of Gs-a have been proposed to arise from alternative splicing of precursor RNA, including proteins of 45 and 52 kD. However the oligonucleotide probe used in the present study should have all of these mRNAs. Thus it would appear that the 45 kD, cholera toxin-sensitive protein present in the striatum is distinct from the Gs-a proteins that have so far been described. D1 receptors may therefore be coupled to adenylyl cyclase by this novel and protein. Supported by grants from the Medical Research Council of Canada and the British Columbia Health Care Research Foundation, S.R.V. is an M.R.C. Scienust. l S.fr.37.6
J
8-Bromo cyclic AMP differentiy regulates astrocytic Go, i proteins depending on the cell culture conditions Malls, M., Glowinski, J. and Pr~mont, J. Chaire de Neuropharmacologie, INSERM UI I4, CollJgede France, 11 place Marcelin Berthelot, 75231 Paris cedex 05, France O proteins (GTP binding proteins) involved in receptor-effector coupling are subject to numerous posttranslational modifications. In particular, a (and fl subunits) oi pertussis-toxin (PTX) - sensitive Go (39 kDa) and Gi (40-41 kDa) proteins linked to inhibition of adenyiate cyclase and opening of some Ca ++ and K + channels seem to be in vitro targets of protein kinases A and C. (Watanabe et al., 1988, FEBS Lett. 236, 372-374; Katada et al., 1985, Eur. J.
A novd Gs prote'm in the stHatum Vincent, S.R., Drinnan, S.L. and Hope, B.T. K'msz~en Laboratoryof NeurologicalResearch, Department of Psychiatry, The University of British Columbi~ Vancouver, B.C., V6T 1WS, Canada
Many hormone and neurotransmitter receptors are coupled to adenylyl cyclase by the GTP-binding protein Gs. In the striatum, activation of D1 dopamine receptors results in a stimulation of adenylyl cyclase. Both the binding of D1 agonists and the stimulation of adenylyl cyclase are dependent upon GTP, implicating Gs in this action. However, the dopamine-stimulated adenylyl cyclase appears to have properties distinct from other agonist-stimulated adenylyl cyclases. Dopamine stimulation is readily observed in both brain homogena~es and slices, while fl-adrenergic stimulation, which can be detected in slices, is lost upon homogenization. This suggests that the D1 and fl-adrenergic ~to~ are coupled to adenylyl cyclase in distinct ways. We have used a synthetic oligonucleotide probe to the cholera toxin ADP-ribosylation site of the alpha subunit of Gs, to det~.t the mRNA for this protein in the rat brain. Northern blot analysis of whole rat brain mRNA indicated that a single mRNA was present of about 1900 bases. This is consistent with findings in other tissues. This message was not detectable in the striation. This probe was also used for the in situ localization of Gs-a mRNA in the rat brain. A regionally selective distribution was found, with high levels in the cortex, hippocampus and cerebellum. Of particular interest was the very low level of signal in the striatum. A closer examination of the distribution of Gs-a mRNA in the forebrain indicated that the regions in which this message was not detectable corresponded to those areas receiving a major dopamine innervation from the midbrain; the caudate-pmamen, nucleus accumbens and oifactory tubercle. Cholera toxin ADP-ribosylates the alpha subunit of Gs, thereby activating this protein. In striatal membranes, cholera toxin results in the ADP-ribosylation of a major band of 45 kD, and a minor band of 52 kD. This is opposite to the pattern of ADP-ribosylated proteins observed in other brain regions. These results indicate that a novel form of Gs-a is present in the striatum where it may act to couple the D1 receptor to adenylyl cyclase. Multiple forms of Gs-a have been proposed to arise from alternative splicing of precursor RNA, including proteins of 45 and 52 kD. However the oligonucleotide probe used in the present study should have all of these mRNAs. Thus it would appear that the 45 kD, cholera toxin-sensitive protein present in the striatum is distinct from the Gs-a proteins that have so far been described. D1 receptors may therefore be coupled to adenylyl cyclase by this novel and protein. Supported by grants from the Medical Research Council of Canada and the British Columbia Health Care Research Foundation, S.R.V. is an M.R.C. Scienust. l S.fr.37.6
J
8-Bromo cyclic AMP differentiy regulates astrocytic Go, i proteins depending on the cell culture conditions Malls, M., Glowinski, J. and Pr~mont, J. Chaire de Neuropharmacologie, INSERM UI I4, CollJgede France, 11 place Marcelin Berthelot, 75231 Paris cedex 05, France O proteins (GTP binding proteins) involved in receptor-effector coupling are subject to numerous posttranslational modifications. In particular, a (and fl subunits) oi pertussis-toxin (PTX) - sensitive Go (39 kDa) and Gi (40-41 kDa) proteins linked to inhibition of adenyiate cyclase and opening of some Ca ++ and K + channels seem to be in vitro targets of protein kinases A and C. (Watanabe et al., 1988, FEBS Lett. 236, 372-374; Katada et al., 1985, Eur. J.