RESEARCH INTERESTS
Mechanism of phototransduction in vertebrate retinal photoreceptors.

Phototransduction is a mechanism to change photon signals to neuronal signals in retinal photoreceptors.  Cyclic GMP is a key component, and change by light of the cGMP level in photoreceptors is the most fundamental reaction in the system.  There are two mechanisms for the alteration of cGMP concentration in photoreceptors; reduction of cGMP concentration by cGMP phosphodiesterase (PDE) and increase of cGMP concentration by guanylate cyclase (GC).  Using biochemical and molecular biological methods, our group has been studied these two aspects of the change of cGMP level in retinal photoreceptors under the support of National Institute of Health, National Eye Institute. 

In the mechanism to reduce cytoplasmic cGMP level in retinal photoreceptors, PDE is a key enzyme. The PDE activation by light is a typical G-protein-dependent signal transduction. Light changes the conformation of rhodopsin (receptor) in photoreceptor outer segment membranes. The illuminated rhodopsin stimulates GTP/GDP exchange on T (transducin, retinal G-protein), followed by dissociation of GTP/Ta (GTP-bound a subunit of T) from Tbg (bg subunits of T). The GTP/Ta activates PDE (effector).  We have studied this mechanism using both amphibian and mammalian retinal photoreceptors. We have revealed (1) regulation of T subunits, (2) functions of PDE subunits, (3) protein-protein interactions involved in the regulatory mechanism of PDE by Ta and (4) regulation of the interactions by protein modifications (phosphorylation and ADP-ribosylation).  Very recently, we proposed a new mechanism for the turnoff of PDE activation; deactivation of GTP/Ta -activated PDE without GTP hydrolysis.  We found that a PDE subunit (g ) complexed with GTP/Ta is phosphorylated by cyclin-dependent protein kinase 5 (Cdk5), and that the phosphorylated subunit is released from GTP/Ta and return to the PDE catalytic subunits.  It has been believed that Cdk5 is involved in various neuronal degenerative diseases such as Alzheimer’s disease.  Thus, these studies also suggest the new possibility that Cdk5 may also be involved in retina degeneration. 

In order to increase cytoplasmic cGMP in retinal photoreceptors, we have focused to reveal the activation mechanism of retinal guanylate cyclase (retGC). We first identified retGC in frog, bovine and toad retinas. Then we found a new signal transduction pathway for retGC activation. We introduced the effects of illuminated rhodopsin and ATP into the retGC activation by GC-activating proteins (GCAPs). We have shown that illuminated rhodopsin is the signal for ATP binding to retGC and the ATP binding is essential for large activation of retGC by GCAPs. These results are totally new and the model based on these results clearly different from the current model. This model will be helpful to understand the mechanism for retGC regulation and to explain retinal abnormalities due to mutations of proteins involved in the retGC/GCAP system. 

Selected publications: 

• Yamazaki, A., Sen, I., Bitensky, M.W., Casnelli, J. and Greengard, P. Cyclic GMP specific, high affinity, noncatalytic binding sites on light activated phosphodiesterase. J. Biol. Chem., 255: 11619-11624, 1980. 
• Yamazaki, A., Bartucca, F., Ting, A. and Bitensky, M.W. Reciprocal effects of an inhibitory factor on catalytic activity and noncatalytic cGMP binding sites of rod phosphodiesterase. Proc. Natl. Acad. Sci. USA., 79: 3702-3706, 1982. 
• Yamazaki, A., Stein, P.J., Chernoff, N. and Bitensky, M.W. Activation mechanism of rod outer segment cycle GMP phosphodiesterase: Release of inhibitor by the GTP/GTP-binding protein. J. Biol. Chem., 258: 8188-8194, 1983. 
• Yamazaki, A., Tatsumi, M., Torney, D.C. and Bitensky, M.W. GTP-binding protein in rod outer segments: (1) The role of each subunit in the GTP hydrolytic cycle. J. Biol. Chem., 262: 9316-9323, 1987. 
• Yamazaki, A., Bitensky, M.W. and Garcia-Sainz, J.A. GTP-binding protein in rod outer segments: (2) An essential role for Mg2+ in signal amplification. J. Biol. Chem., 262: 9324-9331, 1987. 
• Yamazaki, A., Hayashi, F., Tatsumi, M., Bitensky, M.W. and George, J.S. Interactions between the subunits of transducin and cyclic GMP phosphodiesterase in Rana catesbina rod photoreceptors. J. Biol. Chem., 265: 11539-11548, 1990. 
• Hayashi, F., Lin, G.Y., Matsumoto, H. and Yamazaki, A. Phosphatidylinositol-stimulated phosphorylation of an inhibitory subunit of cGMP phosphodiesterase in vertebrate rod photoreceptors. Proc. Natl . Acad. Sci. USA., 88: 4333-4337, 1991. 
• Hayashi, F. and Yamazaki, A. Polymorphism in purified guanylate cyclase from vertebrate rod photoreceptors. Proc. Natl . Acad. Sci. USA., 88: 4746-4750, 1991. 
• Umbarger, K.O., Yamazaki, M., Hutson, L.D., Hayashi, F. and Yamazaki, A. Heterogenity of transducin, Retinal G-protein, from frog photoreceptors: Biochemical identification and characterization of new subunits. J. Biol. Chem., 267: 19494-19502, 1992. 
• Yamazaki, A., Yamazaki, M., Tsuboi, S., Kishigami, A., Umbarger, K.O., Hutson, L.D., Madland, W.T.and Hayashi, F. Regulatory G-protein by an effector in the GTP-dependent signal transducin: An inhibitory subunit of cGMP phosphodiesterase inhibits GTP hydrolysis by transducin in vertebrate rod photoreceptors. J. Biol. Chem., 268: 8899-8907, 1993. 
• Tsuboi, S., Matsumoto, H., Jackson, K. W., Tsujimoto, K., Williams, T., and Yamazaki, A. Phosphorylation of an inhibitory subunit of cGMP phosphodiesterase in Rana catesbiana photoreceptor, (1) characterization of phosphorylation. J. Biol. Chem., 269, 15016 - 15023, 1994. 
• Tsuboi, S., Matsumoto, H., and Yamazaki, A. Phosphorylation of an inhibitory subunit of cGMP phosphodiesterase in Rana catesbiana photoreceptor, (2) A possible mechanism for the turnoff of cGMP phosphodiesterase without GTP hydrolysis. J. Biol. Chem., 269, 15024 - 15029, 1994. 
• Yamazaki, A., Bondarenko, V.A., Dua, S., Yamazaki, M., Usukura, J., and Hayashi, F.  Possible stimulation of retinal rod reconvery to dark state by cGMP release from cGMP phosphodiesterase noncatalytic sites. J. Biol. Chem. 271, 32495-32498, 1996.
• Bondarenko, V.A., Desai, M., Dua, S., Yamazaki, M., Amin, R.H., Kinumi, T., Ohashi, M.  Komori, N., Matsumoto, H., Jackson, K.W., Hayashi, F., Usukura, J., Lipkin, V.M. and Yamazaki, A. Endogenous ADP-ribosylation of the inhibitory subunit of cGMP phosphodiesterase in rod photoreceptors. J. Biol. Chem. 272, 15856-15864, 1997. 
• Seno, K., Kishigami, A., Ihara, S., Maeda, T., Bondarenko, V.A., Nishizawa, Y., Usukura, J., Yamazaki, A., and Hayashi, F.  A possible role of RGS9 in phototransduction: A bridge between the cGMP-phosphodiesterase system and the guanylyl cyclase system.  J. Biol. Chem,  273: 22169 - 22172,  1998.
• Yu, H., Olshevskaya, E., Duda, T., Seno, K., Hayashi, F., Sharm, R. K., Dizhoor, A. M. and  Yamazaki, A.  Activation of retinal guanylyl cyclase-1 by Ca2+ -binding proteins involved its dimerization.  J. Biol. Chem.  274: 15547 - 1555, 1999. 
• Matsuura, I., Bondarenko, V.A., Maeda, T., Kachi. S., Yamazaki, M., Usukura, J.,  Hayashi, F. and Yamazaki, A. Phosphorylation of cyclin-dependent protein-kinase 5 of the regulatory subunit of retinal cGMP phosphodiesterse: 1 Identification of the kinase and its role in the GTP hydrolysis-independent turnoff of phosphodiesterase in vitro. J. Biol. Chem. 275, 32950-32957, 2000.
• Hayashi, F., Matsuura, I., Kachi, S., Maeda, T., Yamamoto, M., Fujii, Y., Yamazaki, M., Usukura, J., and Yamazaki, A. Phosphorylation of cyclin-dependent protein-kinase 5 of the regulatory subunit of retinal cGMP phosphodiesterse: 2 Its role in the GTP hydrolysis-independent turnoff of phosphodiesterase in living photoreceptors. J. Biol. Chem. 275, 32958-32965, 2000. 
• Yamazaki, A., Moskvin, O., and Yamazaki, R. K.  Phosphoryaltion by cyclin-dependent protein kinase 5 of the regulatory subunit (Pg) of retinal cGMP phosphodiesterase (PDE6): Its implications in phototransduction.  In: Calucium and phototransduction, edited by W. Baehr and K. Palczewski, .Adv. Exp. Med. Biol. 514: 131-153,  2002.
• Yamazaki, M., Ning, L., Bondarenko, V. A., Yamazaki, R. K., Baehr, W., and Yamazaki, A.  Binding of cGMP to GAF domains in amphibian rod photoreceptor cGMP phosphodiesterase (PDE): Identification of GAF domains in PDE ab subunit and distinct domains in the PDE g subunit involved in the stimulation of cGMP binding to GAF domains. J. Biol. Chem. 277: 40675-40686,  2002.
• Kajimura, N., Yamazaki, M., Morikawa, K., Yamazaki, A., and Mayanagi, K. Three-dimensional structure of the non-activated form of cGMP phosphodiesterase 6 and comparison of its average image with those of activated forms.  Journal Structural Biology, 139: 27-38, 2002. 
• Yamazaki, A., Yu, H., Yamazaki, M., Honkawa, H., Usukura, J., and Yamazaki, R. K.  A critical role for ATP in the stimulation of guanylyl cyclase by guanylyl cyclase-activating proteins. J. Biol. Chem. 278: 33150-33160, 2003.
• Yamazaki M, Usukura J, Yamazaki RK, Yamazaki A. ATP binding is required for physiological activation of retinal guanylate cyclase.
  Biochem Biophys Res Commun. 2005 Dec 16;338(2):1291-8.
• Yamazaki A, Yamazaki M, Yamazaki RK, Usukura J.  Illuminated rhodopsin is required for strong activation of retinal guanylate cyclase by guanylate cyclase-activating proteins.
  Biochemistry. 2006 Feb 14;45(6):1899-909.