Rapid and Reproducible Deactivation of Rhodopsin Requires Multiple Phosphorylation Sites

Efficient single-photon detection by retinal rod photoreceptors requires timely and reproducible deactivation of rhodopsin. Like other G protein-coupled receptors, rhodopsin contains multiple sites for phosphorylation at its COOH-terminal domain. Transgenic and electrophysiological methods were used to functionally dissect the role of the multiple phosphorylation sites during deactivation of rhodopsin in intact mouse rods. Mutant rhodopsins bearing zero, one (S338), or two (S334/S338) phosphorylation sites generated single-photon responses with greatly prolonged, exponentially distributed durations. Responses from rods expressing mutant rhodopsins bearing more than two phosphorylation sites declined along smooth, reproducible time courses; the rate of recovery increased with increasing numbers of phosphorylation sites. We conclude that multiple phosphorylation of rhodopsin is necessary for rapid and reproducible deactivation.

[1]  R S Johnson,et al.  Characterization of a truncated form of arrestin isolated from bovine rod outer segments , 1994, Protein science : a publication of the Protein Society.

[2]  David J. Baylor,et al.  Mechanisms of rhodopsin inactivation in vivo as revealed by a COOH-terminal truncation mutant , 1995, Science.

[3]  E. Dratz,et al.  Phosphorylation at sites near rhodopsin'scarboxyl-terminus regulates light initiated CGMP hydrolysis , 1984, Vision Research.

[4]  D. Baylor,et al.  Responses of retinal rods to single photons. , 1979, The Journal of physiology.

[5]  T. Lamb,et al.  Amplification and kinetics of the activation steps in phototransduction. , 1993, Biochimica et biophysica acta.

[6]  E. Weiss,et al.  Rhodopsin Phosphorylation Sites and Their Role in Arrestin Binding* , 1997, The Journal of Biological Chemistry.

[7]  K. Yau,et al.  Guanosine 3',5'‐cyclic monophosphate‐activated conductance studied in a truncated rod outer segment of the toad. , 1988, The Journal of physiology.

[8]  D. Baylor,et al.  The photocurrent, noise and spectral sensitivity of rods of the monkey Macaca fascicularis. , 1984, The Journal of physiology.

[9]  D. Baylor,et al.  Two components of electrical dark noise in toad retinal rod outer segments. , 1980, The Journal of physiology.

[10]  R. Crouch,et al.  Mass spectrometric identification of phosphorylation sites in bleached bovine rhodopsin. , 1993, Biochemistry.

[11]  A. Hodgkin,et al.  Detection and resolution of visual stimuli by turtle photoreceptors , 1973, The Journal of physiology.

[12]  J B Hurley,et al.  Abnormal photoresponses and light-induced apoptosis in rods lacking rhodopsin kinase. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[13]  U Wilden,et al.  Duration and amplitude of the light-induced cGMP hydrolysis in vertebrate photoreceptors are regulated by multiple phosphorylation of rhodopsin and by arrestin binding. , 1995, Biochemistry.

[14]  H. Khorana,et al.  Structural features of the C-terminal domain of bovine rhodopsin: a site-directed spin-labeling study. , 1999, Biochemistry.

[15]  M. Chabre,et al.  Interaction between photoexcited rhodopsin and peripheral enzymes in frog retinal rods. Influence on the postmetarhodopsin II decay and phosphorylation rate of rhodopsin. , 1983, European journal of biochemistry.

[16]  K. Palczewski,et al.  Substrate recognition determinants for rhodopsin kinase: studies with synthetic peptides, polyanions, and polycations. , 1989, Biochemistry.

[17]  K. Palczewski,et al.  Sequential phosphorylation of rhodopsin at multiple sites. , 1993, Biochemistry.

[18]  H. Khorana,et al.  Structure and function in rhodopsin: peptide sequences in the cytoplasmic loops of rhodopsin are intimately involved in interaction with rhodopsin kinase. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[19]  S. W. Hall,et al.  Phosphodiesterase activation by photoexcited rhodopsin is quenched when rhodopsin is phosphorylated and binds the intrinsic 48-kDa protein of rod outer segments. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[20]  K. Palczewski,et al.  Anti-rhodopsin monoclonal antibodies of defined specificity: Characterization and application , 1991, Vision Research.

[21]  R. Molday,et al.  Monoclonal antibodies to rhodopsin: characterization, cross-reactivity, and application as structural probes. , 1983, Biochemistry.

[22]  R L Sidman,et al.  Morphological, physiological, and biochemical changes in rhodopsin knockout mice. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[23]  B. Hogan,et al.  Manipulating the mouse embryo: A laboratory manual , 1986 .

[24]  T. Lamb,et al.  Variability in the Time Course of Single Photon Responses from Toad Rods Termination of Rhodopsin’s Activity , 1999, Neuron.

[25]  K. Palczewski,et al.  Structural and Enzymatic Aspects of Rhodopsin Phosphorylation (*) , 1996, The Journal of Biological Chemistry.

[26]  L. Lagnado,et al.  G-protein deactivation is rate-limiting for shut-off of the phototransduction cascade , 1997, Nature.

[27]  T. Dryja,et al.  Transgenic mice carrying the dominant rhodopsin mutation P347S: evidence for defective vectorial transport of rhodopsin to the outer segments. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[28]  U. Wolfrum,et al.  Rhodopsin’s Carboxy-Terminal Cytoplasmic Tail Acts as a Membrane Receptor for Cytoplasmic Dynein by Binding to the Dynein Light Chain Tctex-1 , 1999, Cell.

[29]  P. Sieving,et al.  The electroretinogram of the rhodopsin knockout mouse , 1999, Visual Neuroscience.

[30]  A. Milam,et al.  Rhodopsin Phosphorylation and Dephosphorylation in Vivo(*) , 1995, The Journal of Biological Chemistry.

[31]  R. Hodges,et al.  Antigen-antibody interaction. Synthetic peptides define linear antigenic determinants recognized by monoclonal antibodies directed to the cytoplasmic carboxyl terminus of rhodopsin. , 1988, The Journal of biological chemistry.

[32]  P. Hargrave,et al.  A monoclonal antibody specific for the phosphorylated epitope of rhodopsin: comparison with other anti-phosphoprotein antibodies. , 1988, Hybridoma.

[33]  K. Hofmann,et al.  A model for the recovery kinetics of rod phototransduction, based on the enzymatic deactivation of rhodopsin. , 1998, Biophysical journal.

[34]  J. Findlay,et al.  Phosphorylation of ovine rhodopsin. Identification of the phosphorylated sites. , 1984, The Biochemical journal.

[35]  J. Hurley,et al.  Rhodopsin phosphorylation and its role in photoreceptor function , 1998, Vision Research.

[36]  Denis A. Baylor,et al.  Prolonged photoresponses in transgenic mouse rods lacking arrestin , 1997, Nature.

[37]  K. Palczewski,et al.  Functional differences in the interaction of arrestin and its splice variant, p44, with rhodopsin. , 1997, Biochemistry.

[38]  C. Chothia.  The nature of the accessible and buried surfaces in proteins. , 1976, Journal of molecular biology.

[39]  J L Benovic,et al.  Phosphorylation/dephosphorylation of the beta-adrenergic receptor regulates its functional coupling to adenylate cyclase and subcellular distribution. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[40]  A. Milam,et al.  A splice variant of arrestin. Molecular cloning and localization in bovine retina. , 1994, The Journal of biological chemistry.

[41]  K. Palczewski,et al.  Mechanism of rhodopsin kinase activation. , 1991, The Journal of biological chemistry.

[42]  P. Hargrave,et al.  Phosphorylation sites in bovine rhodopsin. , 1993, Biochemistry.

[43]  G. Rose,et al.  Hydrophobicity of amino acid residues in globular proteins. , 1985, Science.

[44]  U Wilden,et al.  Light-dependent phosphorylation of rhodopsin: number of phosphorylation sites. , 1982, Biochemistry.

[45]  E. Weiss,et al.  Rhodopsin Mutants Discriminate Sites Important for the Activation of Rhodopsin Kinase and G(*) , 1995, The Journal of Biological Chemistry.

[46]  Bovine Retina,et al.  A Splice Variant of Arrestin , 1994 .

[47]  D. Baylor,et al.  Origin of reproducibility in the responses of retinal rods to single photons. , 1998, Biophysical journal.

[48]  K. Palczewski,et al.  Control of rhodopsin multiple phosphorylation. , 1994, Biochemistry.

[49]  Jeannie Chen,et al.  [11] Functional study of rhodopsin phosphorylation in vivo , 2000 .

[50]  W. Dreyer,et al.  Light dependent phosphorylation of rhodopsin by ATP , 1972, FEBS letters.

[51]  K. Palczewski,et al.  G-protein-coupled receptor kinases. , 1991, Trends in biochemical sciences.

[52]  K. Hofmann,et al.  Interaction between photoactivated rhodopsin and its kinase: stability and kinetics of complex formation. , 1993, Biochemistry.