Molecular

determinants

signal transduction Daniel Brandeis

Site-directed wealth

University,

mutagenesis

of information

determination

of the spectral in activation

acid substitutions cell culture

of the three attack

human

pigments

Massachusetts,

amino

acid

properties

found

now

and function

with

been

has provided

responsible

retinitis

the amino

pigmentosa.

established

opening

the way for a similar proteins.

1992, 2:428-432

The human retina contains three other visual pigments in addition to rhodopsin. These are the blue, green, and red color vision pigments of cone photoreceptor cells. Whereas the application of modern techniques of molecular biology is of enormous benefit to the study of the structure and function of rhodopsin, it is essential to the study of the human color vision pigments. This is simply a reflection of the fact that the rod pigment rhodopsin may be obtained in large quantities from cattle retina, whereas human color vision pigments can be obtained in biochemically useful quantities only from heterologous expression systems.

Rhodopsin absorbs light maximally at 500 nm. Upon absorption of light, the 11 -c&retinal chromophore isomerizes to the all-transform, initiating a series of events leading ultimately to closure of Na’ channels in the plasma membrane and hyperpolarization of the rod cell. The initial response is controlled by a conformational change in rhodopsin resulting from photoisomerization of the chromophore in the buried binding pocket of the protein. A well characterized sequence of spectra1 intermediates follows the absorption of a photon of light by the protein [7]. One intermediate in particular, metarhodopsin II, has been shown to be the active species capable of activating the G protein, transducin [S]. Formation of metarhodopsin II from the preceding intermediate metarhodopsin I is accompanied by loss of the proton from the Schiff base linkage, resulting in a large shift of absorption maximum from 478 nm to 380 nm [9]. Metarhodopsin II is the only active intermediate. Upon its decay, the all-truns-retinal chromophore is released from the protein leaving opsin, which is com-

In this review, recent studies are highlighted in which molecular biological approaches have been used to study the mechanism of wavelength regulation in the visual pigments, the involvement of rhodopsin mutations in retinitis pigmentosa, and the mechanism of activation and inactivation of rhodopsin.

Wavelength

regulation

-

@ Current

in the visual pigments

One of the challenging problems for studies of visual systems is to elucidate the mechanism of wavelength regulation in the visual pigments. In free solution, a Schiff base of ll-&retinal has an absorption maximum of about 355nm. Upon binding to opsin the spectrum of the chromophore undergoes a shift to the red such that the pigment absorbs maximally at 500 nm. Similarly, the three color vision pigments contain an identical ll-cis-

Abbreviations

428

In

for expression

pletely inactive. Clearly, the elucidation of the structure of metarhodopsin II is central to an understanding of the mechanisms of phototransduction.

Rhodopsin, the visual pigment of rod photoreceptor cells, is a member of the large family of GTP-binding protein (G-protein)-linked receptors [ 1,2*,3,4*,5,6*]. Rhodopsin is composed of a polypeptide chain opsin, and a covalently bound 1 l-&retinal chromophore. As is typical of other G-protein-linked receptors, rhodopsin is a membrane protein, embedded within the lipid bilayer as a helical bundle with seven transmembrane a-helical segments. The retinal chromophore is bound to the protein within the membrane embedded region by means of a protonated Schiff base linkage to the E-amino group of ~~~296. ~~~296 is located in the seventh transmembrane segment.

Endo H-endoglycosidase

a

for the

and the effect

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in Neurobiology

Introduction

rhodopsin

residues

of the protein,

in patients

have

USA

of the chromophore,

color vision pigments,

Opinion

and

D. Oprian

and inactivation

systems

on the structure

Current

in the visual

of the visual pigment

acids involved addition,

properties

Waltham,

regarding

of amino

of spectral

H; C protein2331.

6.

OP~UANDD: The Ligand Binding Domain of Rhodopsin and J Biomembr Bioenerg Other G-Protein-Linked Receptors. 1992, 24:211-217. A summary of recent chemical modification and mutagenesis studies which suggest that the ligand-binding domains of the G~protein-linked receptors are highly similar to each other. .

7.

WAU) G: Molecular

Basis of Visual Excitation.

Science 1968,

162:230-239.

8.

9.

EMEISD, KUHN H, REICHERTJ, HOFMANNKP: Complex Formation Between Metarhodopsin II and GTP-Binding Protein in Bovine Photoreceptor Membranes Leads to a Shift of the Photoproduct Equilibrium. FEBS Lett 1982, 143:23-34. KG, HUBHAKDR, BROWN PK, WALD G: Tautomeric of Metarhodopsin. J Gen Physiol 1963, 47:215-240.

MATIHEWS

Forms 10.

ZHUKOVSKYEA, OPRIAN DD: Effect of Carboxylic Acid Side Chains on the Absorption Maximum of Visual Pigments. Science 1989, 246:92%930.

11.

SAKMARTP, FRANKE RR, KHORANA HG: Glutamic Acid-l 13 Serves as the Retinylidene SchitT Base Counterion in Bovine Rhodopsin. Proc Nat1 Acad Sci IJSA 1989, 86:8309-8313.

12.

NATHANSJ: Determinants of Visual Pigment Absorbance: Identification of the Retinylldene Schtis Base Counterion in Bovine Rhodopsin. Biocbemisty 1990, 29~97469752.

13.

RADDING CR, WALD G: Acid-Base Properties and Opsin. J Gen Pbysiol 1956, 30:903-923.

14.

UN SW, SAKMARTP, FRANKERR, KHORANAHG, MATHIES RA: Resonance Raman Microprobe Spectroscopy of Rhodopsin Mutants: Effects of Substitutions of Carboxyl Groups in the Third Transmembrane Helix. Biop@sJ 1992, 61:A8 (45).

15.

Genetics of NATHANSJ, THOMAS D, HOGNESS D: Molecular Human Color Vision: the Genes Encoding Blue, Green, and Red Pigments. Science 1986, 232:193%202.

16.

NEITZ M, NEITZ J, JACOBS GH: Spectral Tuning of Pigments Underlying Red-Green Color Vision. Science 1991, 252~971-974.

17.

KOSOWZR EM: Assignment of Groups Responsible for the ‘Opsin Shift’ and Light Absorptions of Rhodopsin and Red, Green, and Blue Iodopsins (Cone Pigments). Proc Nat1 Acud Sci USA 1988, 85:10761080.

18.

OPR~ANDD, ASENJOA&, LEE N, PELLETIER SL: Design, Chemical Synthesis, and Expression of Genes for the Three Human Color Vision Pigments. Biochemisfy 1991, 30:11367-11372.

Note added in proof Merbs and Nathans (Nature 1992, 356:433-435) also reported expression of the human color genes.

transduction

1.

studies on the structure

The Lys296 mutants are of interest not only for the insight they provide into the mechanism of activation/inactivation of rhodopsin, but also because of their relevance to a mutation found in a family with retinitis pigmentosa. Keen et al. [21] have described a family from England with this disease in which the mutation in the rhodopsin gene is a change of ~~~296 to Glu. The family is distinguished from others by having a severe form of the disease with early onset and the appearance of cataracts in the third or fourth decade of life. Robinson et al. [29] prepared the mutant K296E and found that it was constitutively active in in vitro assays with transducin, as were the other mutants with changes at position 296. This observation raises the possibility that degeneration of the photoreceptor cells in patients with this mutation stems from persistent activation of the signalling pathway. This is the first retinitis pigmentosa mutant for which a defect has been documented in the signal transduction pathway.

and signal

of Rhodopsin

431

432

Sensory

systems

19.

DARTNAU HJA, BOWMAKER JK, MOLLON

20.

DR~JA TP, MCGEE TL, REICHEL E, HAITN LB, COWIE~ GS, YANDEU DW, SANDBERG MA, BERSON EL: A Point Mutation of Rhodopsin Gene in One Form of Retinitis Pigmentosa. Nub-e 1990, 343:364-366.

21.

JD: Human Visual Pigments: Microspectrophotometric Results from the Eyes of Seven Persons. PYOC R Sac Lond [Biol] 1983, 220:115-130.

KEEN

TJ, INGLFIHEARNCF, IESTER DH,

BASHIR R, JAY M, BIRD AC,

JAY B, BHATTACHARYA SS:

Autosomal Dominant Retinitis Pigmentosa: Four New Mutations in Rhodopsin, One of Them in the Retinal Attachment Site. Genomics 1991, 11:19’%205.

22.

MCWILL~AM P, FARRQ GJ, HENNA P, BRADLEY DG, HUMPHKIES MM, SHAW EM, MCCONNELL DJ, LAWER M, SHEILS D, RYAN C, ~7 AL.: Autosomal Dominant Retinitis Pigmentosa (ADRP): Localization of an ADRP Gene to the Long Arm of Chromosome 3. Genomics 1989, 5:619-622.

23.

DR~JA

24.

DRVA

25.

TP, ILWN LB, McGek~ TL, COWLEYGS, BFRSON EL: Mutation Spectrum of the Rhodopsin Gene Among Patients with Autosomal Dominant Retinitis Pigmentosa. IVZI~Opbthal Vis Sci 1991, 32:890. TP,

Gores P, NATHANS J: Rhodopsin Mutations in Autosomal Dominant Retinitis Pigmentosa. Proc: Nutl Acud Sci USA1991, 88:6481b6485. SUNG CH, SCHNEIDER BG, AGAPWAL N, PAPERMASTER DS, NATHANSJ: Functional Heterogeneity of Mutant Rhodopsins Responsible for Autosomal Dominant Retinitis Pigmentosa. Proc Nut1 Acad Sci IJSA 1991, 88:884CS8844. Mutant rhodopsin genes from retinitis pigmentosa patients were expressed in cell culture. A preliminary characterization of the proteins is given.

17. .

28. .

ZHUKOVSKY

EA, ROBINSON PK, OPK~AN DD: Transducin Activation by Rhodopsin Without a Covalent Bond to the 11 -Cis-Retinal Chromophore. Science 1991, 25 1:55%560. A mutagenesis study showing that the covalent Schiff base linkage of the I l-c&retinal chromophore to rhodopsin is not required for binding the ligand or for activating transducin.

29,

ROBINSON PR, COHEN GB, ZIItJKwsh?l EA, OPRlAN DD: Constitutive Activation of Rhodopsin by Mutation of ~~~296. Rio&s J 1992, 61:A8 (46).

50.

of the L~NGSTAFFC, CN~HOON RD, RANWI RR: Deprotonation Schti Base of Rhodopsin is Obligate in the Activation of the G Protein. Proc Nut1 Acad Sci [ISA 1986, 83:4209-4213.

31.

SECKLER B,

MCGEE

TL, HAYDNLB, COWI~Y GS, OL%ON JE, REICHEL E, SANDBERC MA, BERSON EL: Mutations Within the Rhodopsin Gene in Patients with Autosomal Dominant Retinitis Pigmentosa. N EnglJ A4ed 1990, 323:1302-1307. INGLEZHEAKN CF,

BASHIR

R, LEX’ER DH, JAY M, BIKU AC, A 3-bp Deletion in the Rhodopsin Gene in a Family with Autosomal Dominant Retinitis Pigmentosa. Am J H14m Genet 1991, 48:2630.

RANUO RR: Schiff-Base Deprotonation is MandaLight-Dependent Rhodopsin Phosphorylation. Rio&em J 1989, 264:483493.

tory

for

BILYITACHAKYA SS:

26.

SUNG

CH,

JAC~HS~N

DAVENPORT SG,

CM,

HECKENUWLY

HENNESSEY JR,

JC,

NOWAK~WSKI

MAIIMENEE R,

FISIIW

IH, G,

DI> Oprian, Graduate Ikpartment of Biochemistry, Waltham, Massachusetts 02254, USA.

Brandeis IJniversity,

Molecular determinants of spectral properties and signal transduction in the visual pigments.

Site-directed mutagenesis of the visual pigment rhodopsin has provided a wealth of information regarding amino acid residues responsible for the deter...
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