Scientific		 Protein Pages
Controlling and Supplying the Visual Cascade
Recent update from: 03.07.2006
Controlling and Supplying the Visual Cascade

requires control of light perception, as well as recovery of activated components involved in phototransduction. The pages shown on the right summarise the interactions and dependencies of different proteins involved in human phototransduction. To ease the access we have prepared pages providing information on the following topics:



This page introduces you to the regulatory proteins and biochemistry of the visual cascade.
If you click on a protein on the right, you will receive a description of the chosen step and further pages providing information on that protein can be accessed. 		Arrestin
Protein Kinase C
Recoverin
Rhodopsin Kinase
Protein Phosphatase 2a
Guanylate Cyclase
Phosducin

Regulation Click on image to enlarge
For a high quality copy of the schematics you may inquire at the pagemaster’s office.
Controlling the Trigger

As we heard before metarhodopsin II (META II) formation activates transducin. During bright light illumination META II is phosphorylated by rhodopsin kinase (RHOK) at serine residues 338 and 342 in a ratio of 40:60 respectively (7) During dim light illumination and in the dark META II is phosphorylated by protein kinase C (PRKC) at the C-terminus. Phosphorylation of META II decreases its affinity to GNAT and increases its affinity to Arrestin (SAG). SAG binding prevents interactions of META II and GNAT completely. Inactivated Photopigment exchanges all-trans retinaldehyde with 11-cis retinaldehyde (see further page), detaches SAG and is dephosphorylated by protein phosphatase 2A (PP2A). Recovered photopigment starts phototransduction again with the uptake of a photon (32), (36), (19).

Rhodopsin Rhodopsin Kinase (RHOK)

RHOK is a soluble Ser/ Thr kinase of the retina and pineal gland. In the retina RHOK is found in rods but not in cones. It could be shown that RHOK is attached to the disc membrane (when binding to RHO ) as well as soluble in the cytoplasm (when autophosphorylated). Its N-terminus binds to the third cytoplasmic loop of RHO and phosphorylates it independent of Ca 2+ primarily at Ser residues 343 and 338. RHOK shows autophosphorylation at Thr 489 and Ser488 and is farnesylated at Cys558. Additionally carboxymethylation could be shown. Farnesylation supports membrane attachment and Mg 2+ is a co-factor for full enzymatic activity. Therefore, both are necessary for complete activity on RHO . Autophosphorylation of RHOK reduces phosphorylation of RHO by keeping the RHOK molecule in solution. RHOK activity is controlled by binding to recoverin (RCV1). (4), (13), (14)(20), (28), (29), (31)

Recoverin Recoverin (RCV1)

RCV1 is a Ca 2+ controlled protein with EF-hand Ca 2+ -binding motifs and homology to visinin. At high Ca 2+ concentrations RCV1 is completely loaded with Ca 2+. Ca 2+ loaded RCV1 binds to RHOK . and blocks its activity.
RCV1 is further myristoylated. Myristoylation is necessary for its membrane attachment and induces a cooperative Ca 2+ dependence of RHOK inhibition. Lowered Ca 2+ concentrations cause membrane dissociation of RCV1 , thus releasing the inhibition of RHOK activity. Raised concentrations of RCV1 slowed therefore recovery from photoexcitation by reducing RHO phosphorylation.
RCV1 immunoreactivity could be shown throughout the whole photoreceptor layer of the retina with restrictions to the rods.
Knock-out mice without recoverin gene expression have been established. The missing RCV1 led to a decrease in RHOK activity followed by prolonged PDE activation and impaired adaptation. But ERGs appeared normal. (4), (3), (5), (8), (16), (21), (25)

Arrestin Arrestin (SAG)

Binding of SAG to RHO enhances RHO regeneration by blocking RHO preferential interactions with GNAT and thus providing the possibility to exchange the photobleached chromophore. It associates to both phosphorylated and unphosphorylated META II. Phosphorylation by protein kinase C (PRKC) increases the affinity to RHO . Thus phosphorylation at high Ca2+ concentration in the dark increases SAG activity to enhance RHO regeneration even at dim light. ATP and Ca2+ are components to further enhance its activity. SAG dephosphorylation is established by calcineurin. During light phase SAG localises mostly in outer segments, while during dark phase they can be found mostly in inner segments, nuclei and synaptic termini
SAG knock-out mice have been shown to be affected only in the homozygous state. These knock-outs show very slow recovery of rod responses but not cone responses. (2), (9), (10), (12), (24), (27) (34), (36)

Protein Protein phosphatase 2A (PP2A)

Protein phosphatase 2A (PP2A) is a heterotrimeric protein made of two catalytic and regulatory subunit. It specifically dephosphorylates Ser and Thr residues. A substrates for PP2A are photopigments and Phosducin . photopigments are dephosphorylated to enter the regeneration cycles. Dephosphorylated Phosducin blocks GNB-GNG from rejoining with GNAT to be reactivated. (15), (19), (30)

Protein Protein Kinase C (PRKC)

PRKC belongs to a family of protein kinases with several isoforms phosphorylating Ser and Thr residues. PRKC isoforms are expressed in several different tissues. While isoform alpha is present as a peripheral membrane protein in ROS and IPL PRKCB isoforms are reported to be present in the human RPE.
PRKC shows autophosphorylation. It is activated by phosphatidylinositol and phosphatidylserine together with Ca2+ and a possible influence of the lipid bilayer of membranes. The affinity to these activators is increased by diacylglycerol. Isoforms PRKCD, PRKCE, and PRKZ show no dependence to Ca2+.
PRKC phosphorylates Ser and Thr residues in a set of proteins that are carriers of major functions in phototransduction. The organic phosphate for this activity is taken from ATP and delivered to targets like SAG , and the photopigments to regulate the trigger, and CNCG1 to control the nerve impulse generation. PRKC phosphorylates PDEG for control of the amplification cascade. Photopigment phosphorylation by PRKC takes place at dim light and darkness and is increased by phorbol myristate. The activity is independent of the activation state of the photoreceptor. Even opsin is phosphorylated by PRKC.
The phosphorylation site on rhodopsin is not equal to that of RHOK. PRKC may inhibit RHOK activity on the photopigment.. Solubilised PDEG is phosphorylated at Thr35 which decreases the binding of GNAT and increases the ability to inhibit PDEA-PDEB. PRKC phosphorylates CNCG1 to control the channel together with protein kinase A (PKA) and is therefore the major protein that influences the control of phototransduction . PKA is not abundant to retinal and subretinal tissues. Thus PKA can not be considered as candidate for a retina specific phenotype while PRKCG has been reported to be envolved in RP11. (1), (6), (11), (23), (26), (33), (34), (35)


Controlling Click on image to enlarge
For a high quality copy of the schematics you may inquire at the pagemaster’s office.
Controlling the Amplification Step

PhosducinPhosducin (33-kDa protein, PDC)

An additional protein that regulates the activity of phototransduction is PDC , a cytoplasmic protein abundant in the retina (rods and cones) and pineal gland that binds tightly to GNB-GNG dimers. A prerequisite of GNB-GNG -binding is the atachment of a farnesyl group which mediates membrane association to get into contact with GNB-GNG. This way it inhibits GTP and cGMP hydrolysis by GNAT because rebinding of GNAT to GNB-GNG dimers, and GTP / cGMP exchange for reactivation of GNAT is blocked. Deactivated GNAT cannot bind to PDEG2 and activation of PDEA-PDEB is blocked, too. Thus PDC is a negative regulator in phototransduction.
PDC itself is negatively regulated by phosphorylation. PKA phosphorylates it at Ser73, while protein phosphatase 2A has a dephosphorylating function on PDC . Phosphorylation is high in the dark when there is less turnover in the visual cascade.
Phospholipase 2A is another target of PDC activity. Therefore, PDC reacts on PRKC because phospholipase 2A produces phosphatidyl fatty acids necessary for PRKC stimulation. (15), (17), (18), (22)

References
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    Link Goto Top
  2. Bennett,N. and Sitaramayya,A. Inactivation of photoexcited rhodopsin in retinal rods: the roles of rhodopsin kinase and 48-kDa protein (arrestin). 1988; Biochemistry. 27: 1710-1715. Link Goto Top
  3. Calvert,P.D., Klenchin,V.A., and Bownds,M.D. Rhodopsin Kinase Autophosphorylation Regulates It's Sensivity To Calcium-Recoverin. 1995; Invest.Ophthalmol.Vis.Sci. 36: S269 Goto Top
  4. Chen,C.K., Inglese,J., Lefkowitz,R.J., and Hurley,J.B. Ca(2+)-dependent interaction of recoverin with rhodopsin kinase. 1995; J.Biol.Chem. 270: 18060-18066. Link Goto Top
  5. Chen,Z., Prasad,S., and Cynader,M.S. Establishment of cDNA libraries of twelve different human ocular tissues. 1995; Invest.Ophthalmol.Vis.Sci. Suppl.: S123 Goto Top
  6. Coussens,L., Parker,P.J., Rhee,L., Yang Feng,T.L., Chen,E., Waterfield,M.D., Francke,U., and Ullrich,A. Multiple, distinct forms of bovine and human protein kinase C suggest diversity in cellular signaling pathways. 1986; Science. 233: 859-866.
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  7. Crouch,R.K., Goletz,P., Papac,D., and Knapp,D. Rhodopsin phosphorylation sites. 1994; Invest.Ophthalmol.Vis.Sci. 35 (Suppl.): 1461 Goto Top
  8. Dizhoor,A.M., Ray,S., Kumar,S., Niemi,G., Spencer,M., Brolley,D., Walsh,K.A., Philipov,P.P., Hurley,J.B., and Stryer,L. Recoverin: a calcium sensitive activator of retinal rod guanylate cyclase. 1991; Science. 251: 915-918. Link Goto Top
  9. Dodd,R.L., Makino,C.L., Xu,J., Chen,J., Simon,M.I., and Baylor,D.A. Knockout of Arrestin Delays Final shutoff of the Rod Photoresponse. 1996; Invest.Ophthalmol.Vis.Sci. 37: S5 Goto Top
  10. Edwards,S.C., Ellis,D.Z., and Herrera,D.C. Evidence that calcineurin is responsible for arrestin dephosphorylation in the limulus lateral eye. 1993; Invest.Ophthalmol.Vis.Sci. 34 (Suppl.): 1325 Goto Top
  11. Endert,U., Kohler,K., and Zrenner,E. Light and electron microscopic localization of protein kinase C in the chicken retina. 1994; Invest.Ophthalmol.Vis.Sci. 35: 1581 Goto Top
  12. Glitscher,W. and Ruppel,H. Evidence for ATP-ase activity of arrestin from bovine photoreceptors. 1991; FEBS Lett. 282: 431-435. Link Goto Top
  13. Haga,T., Haga,K., and Kameyama,K. G protein--coupled receptor kinases. 1994; J.Neurochem. 63: 400-412. Link Goto Top
  14. Hargrave,P.A. and McDowell,J.H. Rhodopsin and phototransduction: a model system for G protein- linked receptors. 1992; FASEB Journal. 6: 2323-2331.
    Link Goto Top
  15. Ho,Y.K., Loew,A., Blundell,T., and Bax,B. Phosducin Induces A Structural Shift On Transducin gamma? That Internalizes The G Subunit Farnesyl Group. 1998; Invest.Ophthalmol.Vis.Sci. 39: S442 Goto Top
  16. Hurley,J.B., Dizhoor,A.M., Ray,S., and Stryer,L. Recoverin's role: conclusion withdrawn. 1993; Science. 260: 740 Link Goto Top
  17. Jelsema,C.L. Light activation of phospholipase A2 in rod outer segments of bovine retina and its modulation by GTP-binding proteins. 1987; J.Biol.Chem. 262: 163-168.
    Link Goto Top
  18. Jelsema,C.L. and Axelrod,J. Stimulation of phospholipase A2 activity in bovine rod outer segments by the beta gamma subunits of transducin and its inhibition by the alpha subunit. 1987; Proc.Natl.Acad.Sci.U.S.A. 84: 3623-3627.
    Link Goto Top
  19. Jones,T.A., Barker,H.M., da Cruz,S., Mayer Jäckel,R.E., Hemmings,B.A., Spurr,N.K., Sheer,D., Cohen,P.T., da,C., and Mayer Jaekel,R.E. Localization of the genes encoding the catalytic subunits of protein phosphatase 2A to human chromosome bands 5q23-->q31 and 8p12-->p11.2, respectively. 1993; Cytogenet.Cell Genet. 63: 35-41.
    Link Goto Top
  20. Khani,S.C., Nielsen,L., and Vogt,T.M. Biochemical evidence for pathogenicity of rhodopsin kinase mutations correlated with the oguchi form of congenital stationary night blindness. 1998; Proc.Natl.Acad.Sci.U.S.A. 95: 2824-2827.
    Link Goto Top
  21. Kutuzov,M.A., Shmukler,B.E., Suslov,O.N., Dergachev,A.E., Zargarov,A.A., and Abdulaev,N.G. P26-calcium binding protein from bovine retinal photoreceptor cells. 1991; FEBS Lett. 293: 21-24.
    Link Goto Top
  22. Lee,R.H., Ting,T.D., Lieberman,B.S., Tobias,D.E., Lolley,R.N., and Ho,Y.K. Regulation of retinal cGMP cascade by phosducin in bovine rod photoreceptor cells. Interaction of phosducin and transducin. 1992; J.Biol.Chem. 267: 25104-25112. Link Goto Top
  23. Liu,M.Y., Li,J., and Yau,K.W. Phosphorylation of the N-terminal domain of human rod cyclic nucleotide-gated channel by protein kinase c and the cAMP- dependent protein kinase. 1994; Invest.Ophthalmol.Vis.Sci. 35 (Suppl.): 1474 Goto Top
  24. Lyubarsky,A.L., Pugh,E.N., Falsini,B., Valentini,P., and Chen,J. Arrestin Knock-Out Mice As A Model Of Oguchi's Disease. 1998; Invest.Ophthalmol.Vis.Sci. 39: S643 Goto Top
  25. McGinnis,J.F., Stepanik,P.L., Baehr,W., Subbaraya,I., and Lerious,V. Cloning and sequencing of the 23 kDa mouse photoreceptor cell- specific protein. 1992; FEBS Lett. 302: 172-176.
    Link Goto Top
  26. Newton,A.C. and Williams,D.S. Rhodopsin is the major in situ substrate of protein kinase C in rod outer segments of photoreceptors. 1993; J.Biol.Chem. 268: 18181-18186.
    Link Goto Top
  27. Nir,I. and Ransom,N. Ultrastructural analysis of arrestin distribution in mouse photoreceptors during dark/light cycle. 1993; Exp.Eye Res. 57: 307-318. Link Goto Top
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  29. Palczewski,K., Buczylko,J., Kaplan,M.W., Polans,A.S., and Crabb,J.W. Mechanism of rhodopsin kinase activation. 1991; J.Biol.Chem. 266: 12949-12955.
    Link Goto Top
  30. Pallas,D.C., Weller,W., Jaspers,S., Miller,T.B., Lane,W.S., and Roberts,T.M. The third subunit of protein phosphatase 2A (PP2A), a 55- kilodalton protein which is apparently substituted for by T antigens in complexes with the 36- and 63-kilodalton PP2A subunits, bears little resemblance to T antigens. 1992; J.Virol. 66: 886-893.
    Link Goto Top
  31. Schafer,W.R. and Rine,J. Protein prenylation: genes, enzymes, targets, and functions. 1992; Annu.Rev.Genet. 26:209-37.: 209-237. Link Goto Top
  32. Stryer,L. Visual excitation and recovery Visual excitation and recovery. 1991; J.Biol.Chem. 266: 10711-10714.
    Link Goto Top
  33. Takai,Y., Kishimoto,A., Iwasa,Y., Kawahara,Y., Mori,T., and Nishizuka,Y. Calcium-dependent activation of a multifunctional protein kinase by membrane phospholipids. 1979; J.Biol.Chem. 254: 3692-3695.
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  34. Udovichenko,I.P., Cunnick,J., Gonzalez,K., and Takemoto,D.J. Functional effect of phosphorylation of the photoreceptor phosphodiesterase inhibitory subunit by protein kinase C. 1994; J.Biol.Chem. 269: 9850-9856.
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  35. Wolbring,G. and Cook,N.J. Rapid purification and characterization of protein kinase C from bovine retinal rod outer segments. 1991; Eur.J.Biochem. 201: 601-606.
    Link Goto Top
  36. Yamaki,K., Tsuda,M., and Shinohara,T. The sequence of human retinal S-antigen reveals similarities with alpha-transducin. 1988; FEBS Letters. 234: 39-43.
    Link Goto Top

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