Progress 01/01/90 to 12/31/91
Outputs
Receptor-mediated signal transduction is involved in the regulation of importantcellular activities including adenylate cyclase, phototransduction & growth factor action. Specifically, fluorescence spectroscopic approaches have been used to monitor the rhodopsin-catalyzed activation-deactivation cycle of transducin. We have found that GTP binding to the alpha subunit of the retinal G protein is accompanied by about a 2-fold enhancement in the intrinsic (tryptophan) fluorescence. This enhancement is followed by a decay which occurs over a time period of 2-5 min and appears to represent the GTP-hydrolytic step (i.e. deactivation). The rhodopsin-stimulated GTP binding to the alpha subunit is highly dependent on MgCl2 and on the presence of the beta gamma subunit complex. Maximal rhodopsin-stimulated binding can occur when there are 15 alphaT subunits per 1 beta gammaT complex indicating that beta gammaT can act analytically by continually dissociating from, and
re-associating with, multiple alphaT subunits and thereby promoting their activation. Studies have also been performed with an antibody generated against the caboxyl-terminal of the alpha subunit. This antibody blocks rhodopsin-transducin coupling but potentiates alpha subunit-phosphodiesterase interactions. These results suggest that the carboxyl-terminal domain of the alpha subunit contains the receptor binding site & that interactions at this domain enhance the ability of the retinal G protein to couple to the effector enzyme.
Impacts
(N/A)
Publications
- ERICKSON J. and R.A. CERIONE. 1991. Biochemistry 30: 7112-7118.
- PHILLIPS, W.J. and R.A. CERIONE. 1991. Labeling of the beta subunit of transducin with an environmentally-sensitive fluorescent cysteine reagent: A spectroscopic probe for transducin subunit interactions. J.B.C. 266: 11017-11024,.
|
Progress 01/01/89 to 12/30/89
Outputs
Receptor-mediated signal transduction is involved in the regulation of importantcellular activities including adenylate cyclase, phototransduction & growth factor action. Specifically, fluorescence spectroscopic approaches have been used to monitor the rhodopsin-catalyzed activation-deactivation cycle of transducin. We have found that GTP binding to the alpha subunit of the retinal G protein is accompanied by about a 2-fold enhancement in the intrinsic (tryptophan) fluorescence. This enhancement is followed by a decay which occurs over a time period of 2-5 min and appears to represent the GTP-hydrolytic step (i.e. deactivation). The rhodopsin-stimulated GTP binding to the alpha subunit is highly dependent on MgCl2 and on the presence of the beta gamma subunit complex. Maximal rhodopsin-stimulated binding can occur when there are 15 alphaT subunits per 1 beta gammaT complex indicating that beta gammaT can act analytically by continually dissociating from, and
re-associating with, multiple alphaT subunits and thereby promoting their activation. Studies have also been performed with an antibody generated against the carboxyl-terminal of the alpha subunit. This antibody blocks rhodopsin-transducin coupling but potentiates alpha subunit-phosphodiesterase interactions. These results suggest that the carboxyl-terminal domain of the alpha subunit contains the receptor binding site & that interactions at this domain enhance the ability of the retinal G protein to couple to the effector enzyme.
Impacts
(N/A)
Publications
- PHILLIPS WJ, TRUKAWINSKI S, & CERIONE RA. 1989. An antibody-induced enhancement of the transducin-stimulated cyclic GMP phosphodiesterase activity. J. Biol. Chem., 264: 16679-16688.
- GUY PM, KOLAND JG & CERIONE RA. 1990. The rhodopsin-stimulated activation-deactivation cycle of transducin: Kinetics of the intrinsic tryptophan fluorescence of the alpha subunit. Submitted to Biochemistry.
|
Progress 01/01/88 to 12/30/88
Outputs
Receptor-mediated signal transduction is involved in the regulation of importantcellular activities including adenylate cyclase, phototransduction & growth factor action. Specifically, fluorescence spectroscopic approaches have been used to monitor the rhodopsin-catalyzed activation-deactivation cycle of transducin. We have found that GTP binding to the alpha subunit of the retinal G protein is accompanied by about a 2-fold enhancement in the intrinsic (typtophan) fluorescence. This enhancement is followed by a decay which occurs over a time period of 2-5 min and appears to represent the GTP-hydrolytic step (i.e. deactivation). The rhodopsin-stimulated GTP binding to the alpha subunit is highly dependent on MgC12 and on the presence of the beta gamma subunit complex. Maximal rhodopsin-stimulated binding can occur when there are 15 alphaT subunits per 1 beta gammaT complex indicating that beta gammaT can act analytically by continually dissociating from, and
re-associating with, multiple alphaT subunits and thereby promoting their activation. Studies have also been performed with an antibody generated against the carboxyl-terminal of the alpha subunit. This antibody blocks rhodopsin-transducin coupling but potentiates alpha subunit-phosphodiesterase interactions. These results suggest that the carboxyl-terminal domain of the alpha subunit contains the receptor binding site & that interactions at this domain enhance the ability of the retinal G protein to couple to the effector enzyme.
Impacts
(N/A)
Publications
- CERIONE, R.A., KROLL, S., RAJARAM, R., UNSON, C., GOLDSMITH, P. and SPIEGEL, A. 1988. An antibody directed against the carboxyl-terminal decapeptide of the alpha subunit of the retinal GTP binding protein. J. Biol. Chem. 263:9345-9352.
- PHILLIPS, W.J. and CERIONE, R.A. 1988. The intrinsic fluorescence of the alpha subunit of transducin: measurement of receptor-dependent guanine nucleotide exchange. J. Biol. Chem. 263:15498-15505.
- KROLL, S., PHILLIPS, W.J. and CERIONE, R.A. 1988. The regulation of the cyclic GMP phosphodiesterase by the GDP-bound form of the alpha subunit of transducin. J. Biol. Chem., (in-press).
- PHILLIPS, W.J., TRUKAWINSKI, S. and CERIONE, R.A. 1988. An antibody-induced enhancement of the transducin-stimulated cyclic GMP phosphodiesterase activity. Submitted to J. Biol. Chem.
|
Progress 01/01/87 to 12/30/87
Outputs
Receptor-mediated signal transduction is involved in the regulation of importantcellular activities including adenylate cyclase, phototransduction & growth factor action. Specifically, fluorescence spectroscopic approaches have been used to monitor the rhodopsin-catalyzed activation-deactivation cycle of transducin. We have found that GTP binding to the alpha subunit of the retinal G protein is accompanied by about a 2 fold enhancement in the intrinsic (tryptophan) fluorescence. This enhancement is followed by a decay which occurs over a time period of 2-5 min and appears to represent the GTP-hydrolytic step (i.e. deactivation). The rhodopsin-stimulated GTP binding to the alpha subunit is highly dependent on MgCl2 and on the presence of the beta gamma subunit complex. Maximal rhodopsin-stimulated binding can occur when there are 15 alphaT subunits per 1 begt gammaT complex indicating that beta gammaT can act catalytically by continually dissociating from, and
re-associating with, multiple alphaT subunits and thereby promoting their activation. Studies have also been performed with an antibody generated against the carboxyl-terminal of the alpha subunit. This antibody blocks rhodopsin-transducin coupling but potentiates alpha subunit-phosphodiesterase interactions.
Impacts
(N/A)
Publications
- PHILLIPS, W.J. and CERIONE, R.A. 1988. The Intrinsic fluorescence of the alpha subunit of transducin I. Detection of distinct conformational states. Submitted to J. Biol. Chem.
- PHILLIPS, W.J. and CERIONE, R.A. 1988. The intrinsic fluorescence of the alpha subunit of transducin II. Measurement of receptor-dependent guanine nucleotide exchange. Submitted to J. Biol. Chem.
- CERIONE, R.A., KROLL, S., RAJARAM, R., UNSON, C., GOLDSMITH, P. and SPIEGEL, A. 1988. An antibody directed against the carboxyl-terminal decapeptide of the alpha subunit of the retinal GTP binding protein. Submitted to J. Biol. Chem.
|
Progress 01/01/86 to 12/30/86
Outputs
A major line of study being pursued involves the use of reconstitution approaches to delineate the sequence of events (& the specific protein components involved) in growth factor receptor-mediated signal transduction. The epidermal growth factor receptor & the insulin receptor are being used as models in these studies. Methods have been developed for functionally inserting these proteins into phospholipid vesicle systems. These approaches have enabled the construction of 2 component systems comprised of these growth factor receptors & different pure GTP binding proteins to determine whether the activation of a GTP binding protein is an early event in growth factor action. We have identified specific interactions between the insulin receptor & the pertussis toxin sensitive GTP binding proteins Gi (the inhibitory GTP binding protein of the adenylate cyclase system) & Go (a recently discovered GTP binding protein of unknown function). These interactions which are
reflected by an insulin-stimulated phosphorylation of the GTP binding proteins occur subsequent to insulin-stimulated phosphorylation of the receptor itself. Similar studies are now being performed with the epidermal growth factor receptor to determine the nature of possible transducer proteins in the action of EGF.
Impacts
(N/A)
Publications
- KRUPINSKI, J., LAKONISHOK, M., BENOVIC, J. AND CERIONE, R.A. 1987. Insulin-dependent phosphorylation of GTP binding proteins in phospholipid vesicles. J. Biol. Chem., submitted.
|
|