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Subcellular localization of phosducin in rod photoreceptors

Published online by Cambridge University Press:  05 April 2005

JING CHEN ,
TATSURO YOSHIDA ,
KOICHI NAKANO  and
MARK W. BITENSKY
JING CHEN
Affiliation:
Department of Biomedical Engineering, College of Engineering, Boston University, Boston, MA 02215
TATSURO YOSHIDA
Affiliation:
Department of Biomedical Engineering, College of Engineering, Boston University, Boston, MA 02215
KOICHI NAKANO
Affiliation:
Department of Biomedical Engineering, College of Engineering, Boston University, Boston, MA 02215
MARK W. BITENSKY
Affiliation:
Department of Biomedical Engineering, College of Engineering, Boston University, Boston, MA 02215
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Abstract

Phosducin (Pd) is a 28-kD phosphoprotein whose expression in retina appears limited to photoreceptor cells. Pd binds to the β,γ subunits of transducin (Gt). Their binding affinity is markedly diminished by Pd phosphorylation. While Pd has long been regarded as a candidate for the regulation of Gt, the molecular details of Pd function remain unclear. This gap in understanding is due in part to a lack of precise information concerning the total amount and subcellular localization of rod Pd. While earlier studies suggested that Pd was a rod outer segment (ROS) protein, recent findings have demonstrated that Pd is distributed throughout the rod. In this report, the subcellular distribution and amounts of rat Pd are quantified with immunogold electron microscopy. After light or dark adaptation, retinal tissues were fixed in situ and prepared for ultrathin sectioning and immunogold labeling. Pd concentrations were analyzed over the entire length of the rod. The highest Pd labeling densities were found in the rod synapse. Less intense Pd staining was observed in the ellipsoid and myoid regions, while minimal labeling densities were found in the ROS and the rod nucleus. In contrast with rod Gt, no evidence was found for light-dependent movement of Pd between inner and outer segments. There is a relative paucity of Pd in the ROS as compared with the large amounts of Gt found there. This does not support the earlier idea that Pd could modulate Gt activity by controlling its concentration. On the other hand, the presence of Pd in the nucleus is consistent with its possible role as a regulator of transcription. The functions of Pd in the ellipsoid and myoid regions remain unclear. The highest concentration of Pd was found at the rod synapse, consistent with a suggested role for Pd in the regulation of synaptic function.

Keywords

PhosducinTransducinRodImmunogoldElectron microscopy
Type
Research Article
Information
Visual Neuroscience , Volume 22 , Issue 1 , January 2005 , pp. 19 - 25
Copyright
© 2005 Cambridge University Press

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References

REFERENCES

Aitken, A., Jones, D., Soneji, Y., & Howell, S. (1995). 14-3-3 proteins: Biological function and domain structure. Biochemical Society Transactions 23, 605–611.CrossRefGoogle Scholar
Baehr, W., Morita, E.A., Swanson, R.J., & Applebury, M.L. (1982). Characterization of bovine rod outer segment G-protein. Journal of Biological Chemistry 257, 6452–6460.Google Scholar
Barhite, S., Thibault, C., & Miles, M.F. (1998). Phosducin-like protein (PhLP), a regulator of G beta gamma function, interacts with the proteasomal protein SUG1. Biochimica et Biophysica Acta 1402, 95–101.CrossRefGoogle Scholar
Brown, B.M., Carlson, B.L., Zhu, X., Lolley, R.N., & Craft, C.M. (2002). Light-driven translocation of the protein phosphatase 2A complex regulates light/dark dephosphorylation of phosducin and rhodopsin. Biochemistry 41, 13526–13538.CrossRefGoogle Scholar
Danner, S. & Lohse, M.J. (1996). Phosducin is a ubiquitous G-protein regulator. Proceedings of the National Academy of Sciences of the U.S.A. 93, 10145–10150.CrossRefGoogle Scholar
Hajibagheri, M.N. (1999). Electron Microscopy Methods and Protocols. Totowa, New Jersey: Humana Press, pp. 131–134.CrossRef
Hamm, H.E. & Bownds, M.D. (1986). Protein complement of rod outer segments of frog retina. Biochemistry 25, 4512–4523.CrossRefGoogle Scholar
Hebel, R. & Stromberg, M.W. (1976). Anatomy of the Laboratory Rat. Baltimore, Maryland: Williams and Wilkins, pp. 145–146.
Kühn, H., Bennett, N., Michel-Villaz, M., & Chabre, M. (1981). Interactions between photoexcited rhodopsin and GTP-binding protein: Kinetic and stoichiometric analyses from light-scattering changes. Proceedings of the National Academy of Sciences of the U.S.A. 78, 6873–6877.CrossRefGoogle Scholar
Kuo, C.H. & Miki, N. (1989). Translocation of a photoreceptor-specific MEKA protein by light. Neuroscience Letters 103, 8–10.CrossRefGoogle Scholar
Lee, R.H., Brown, B.M., & Lolley, R.N. (1984). Light-induced dephosphorylation of a 33K protein in rod outer segments of rat retina. Biochemistry 23, 1972–1977.CrossRefGoogle Scholar
Lee, R.H., Lieberman, B.S., & Lolley, R.N. (1987). A novel complex from bovine visual cells of a 33,000-dalton phosphoprotein with beta- and gamma-transducin: Purification and subunit structure. Biochemistry 26, 3983–3990.CrossRefGoogle Scholar
Lee, R.H., Whelan, J.P., Lolley, R.N., & McGinnis, J.F. (1988). The photoreceptor-specific 33 kDa phosphoprotein of mammalian retina: Generation of monospecific antibodies and localization by immunocytochemistry. Experimental Eye Research 46, 829–840.CrossRefGoogle Scholar
Lee, R.H., Brown, B.M., & Lolley, R.N. (1990). Protein kinase A phosphorylates retinal phosducin on serine 73 in situ. Journal of Biological Chemistry 265, 15860–15866.Google Scholar
Lee, R.H., Ting, T.D., Lieberman, B.S., Tobias, D.E., Lolley, R.N., & Ho, Y.K. (1992). Regulation of retinal cGMP cascade by phosducin in bovine rod photoreceptor cells. Interaction of phosducin and transducin. Journal of Biological Chemistry 267, 25104–25112.Google Scholar
Margulis, A., Dang, L., Pulukuri, S., Lee, R., & Sitaramayya, A. (2002). Presence of phosducin in the nuclei of bovine retinal cells. Molecular Vision 8, 477–482.Google Scholar
Mayhew, T.M. & Astle, D. (1997). Photoreceptor number and outer segment disk membrane surface area in the retina of rat: Stereological data for whole organ and average photoreceptor cell. Journal of Neurocytology 26, 53–61.CrossRefGoogle Scholar
Nakano, K., Chen, J., Tarr, G.E., Yoshida, T., Flynn, J.M., & Bitensky, M.W. (2001). Rethinking the role of phosducin: Light-regulated binding of phosducin to 14-3-3 in rod inner segments. Proceedings of the National Academy of Sciences of the U.S.A. 98, 4693–4698.CrossRefGoogle Scholar
Obin, M., Lee, B.Y., Meinke, G., Bohm, A., Lee, R.H., Gaudet, R., Hopp, J.A., Arshavsky, V.Y., Willardson, B.M., & Taylor, A. (2002). Ubiquitylation of the transducin betagamma subunit complex. Regulation by phosducin. Journal of Biological Chemistry 277, 44566–44575.Google Scholar
Pagh-Roehl, K., Lin, D., Su, L., & Burnside, B. (1995). Phosducin and PP33 are in vivo targets of PKA and type 1 or 2A phosphatases, regulators of cell elongation in teleost rod inner-outer segments. Journal of Neuroscience 15, 6475–6488.Google Scholar
Roth, D. & Burgoyne, R.D. (1995). Stimulation of catecholamine secretion from adrenal chromaffin cells by 14-3-3 proteins is due to reorganisation of the cortical actin network. FEBS Lett. 374, 77–81.CrossRefGoogle Scholar
Savage, J.R., McLaughlin, J.N., Skiba, N.P., Hamm, H.E., & Willardson, B.M. (2000). Functional roles of the two domains of phosducin and phosducin-like protein. Journal of Biological Chemistry 275, 30399–30407.CrossRefGoogle Scholar
Sokolov, M., Lyubarsky, A.L., Strissel, K.J., Savchenko, A.B., Govardovskii, V.I., Pugh, E.N., Jr., & Arshavsky, V.Y. (2002). Massive light-driven translocation of transducin between the two major compartments of rod cells: A novel mechanism of light adaptation. Neuron 34, 95–106.CrossRefGoogle Scholar
Sokolov, M., Strissel, K.J., Leskov, I.B., Michaud, N.A., Govardovskii, V.I., & Arshavsky, V.Y. (2004). Phosducin facilitates light-driven transducin translocation in rod photoreceptors: Evidence from the phosducin knockout mouse. Journal of Biological Chemistry 279, 19149–19156.CrossRefGoogle Scholar
Thulin, C.D., Howes, K., Driscoll, C.D., Savage, J.R., Rand, T.A., Baehr, W., & Willardson, B.M. (1999). The immunolocalization and divergent roles of phosducin and phosducin-like protein in the retina. Molecular Vision 5, 40.Google Scholar
Thulin, C.D., Savage, J.R., McLaughlin, J.N., Truscott, S.M., Old, W.M., Ahn, N.G., Resing, K.A., Hamm, H.E., Bitensky, M.W., & Willardson, B.M. (2001). Modulation of the G protein regulator phosducin by Ca2+/calmodulin-dependent protein kinase II phosphorylation and 14-3-3 protein binding. Journal of Biological Chemistry 276, 23805–23815.CrossRefGoogle Scholar
von Schantz, M., Szel, A., van Veen, T., & Farber, D.B. (1994). Expression of soluble phototransduction-associated proteins in ground squirrel retina. Investigative Ophthalmology and Visual Science 35, 3922–3930.Google Scholar
Whelan, J.P. & McGinnis, J.F. (1988). Light-dependent subcellular movement of photoreceptor proteins. Journal of Neuroscience Research 20, 263–270.CrossRefGoogle Scholar
Willardson, B.M., Wilkins, J.F., Yoshida, T., & Bitensky, M.W. (1996). Regulation of phosducin phosphorylation in retinal rods by Ca2+/calmodulin-dependent adenylyl cyclase. Proceedings of the National Academy of Sciences of the U.S.A. 93, 1475–1479.CrossRefGoogle Scholar
Yoshida, T., Willardson, B.M., Wilkins, J.F., Jensen, G.J., Thornton, B.D., & Bitensky, M.W. (1994). The phosphorylation state of phosducin determines its ability to block transducin subunit interactions and inhibit transducin binding to activated rhodopsin. Journal of Biological Chemistry 269, 24050–24057.Google Scholar
Yusim, K., Parnas, H., & Segel, L.A. (2000). Theory for the feedback inhibition of fast release of neurotransmitter. Bulletin of Mathematical Biology 62, 717–757.CrossRefGoogle Scholar
Zhu, X. & Craft, C.M. (1998). Interaction of phosducin and phosducin isoforms with a 26S proteasomal subunit, SUG1. Molecular Vision 4, 13.Google Scholar
Zhu, X. & Craft, C.M. (2000a). Modulation of CRX transactivation activity by phosducin isoforms. Molecular and Cellular Biology 20, 5216–5226.Google Scholar
Zhu, X. & Craft, C.M. (2000b). The carboxyl terminal domain of phosducin functions as a transcriptional activator. Biochemical and Biophysical Research Communications 270, 504–509.Google Scholar