The focus is on light transduction in vision. Specifically, we will study the dim light vertebrate photoreceptor, rhodopsin. Three specific long- range goals are proposed. I. Chemical Mechanism of the Transmission of the Signal (Light-catalyzed Retinal Isomerization) from the Seven Helical Bundle (the Membrane Domain) to the Two Extra-membranous Domains. A conformational change in the cytoplasmic domain occurs on illumination of rhodopsin. This change initiates all the biochemistry of the visual transduction. Our goal is to understand the precise chemical nature of this change. II. Mechanisms in Amplification of the Signal. Study of the binding of GDP-transducin to Metarhodopsin II, exchange of GDP by GTP and release of Talpha GTP. This will include detailed mapping of the contacts between rhodopsin and different forms of transducin. III. Biochemistry and Mechanisms of Adaptation to Light. Understanding adaptation is the major ultimate goal. The following overall processes leading to desensitization and termination of visual transduction are not understood: 1, selection of sites of single phosphorylation and/or polyphosphorylations of rhodopsin by rhodopsin kinase (RK); 2, mechanisms of interactions of phosphorylated rhodopsin with arrestin and possibly other proteins as yet unknown; 3, how do these protein/protein interactions pre-empt the binding sites and structures that are necessary for amplification of the signal (Metarhodopsin II- transducin interaction). Can we understand the accompanying conformational changes in the proteins participating in these processes? IV. Experimental Approaches. These would be interdisciplinary, spanning organic chemistry, biochemistry, molecular biology and biophysics (this in collaborations with Biophysical research groups). Approaches involve extensive mutagenesis in the synthetic rhodopsin gene, expression and purification of the expressed mutant proteins, chemical studies of the mutants including inter and intra-molecular crosslinking, using photosensitive and other bifunctional groups. Introduction of one or two cysteine residues at specific sites and their use as handles for chemical and biophysical (fluorescence, EPR) studies. Studies of the sites of the binding sites of RK, transducin and arrestin to Metarhodopsin. Manipulation as well as limitations of the phosphorylation sites in rhodopsin by extensive amino acid replacements, the role of single V/S multiple phosphorylations in arrestin binding. These studies will include on and off rates of binding of transducin, RK and arrestin to Metarhodopsin II and its phosphorylated forms.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM028289-18
Application #
2608774
Study Section
Visual Sciences C Study Section (VISC)
Project Start
1980-12-01
Project End
1999-11-30
Budget Start
1997-12-01
Budget End
1998-11-30
Support Year
18
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Massachusetts Institute of Technology
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
City
Cambridge
State
MA
Country
United States
Zip Code
02139
Ou, Wen-bin; Yi, Tingfang; Kim, Jong-Myoung et al. (2011) The roles of transmembrane domain helix-III during rhodopsin photoactivation. PLoS One 6:e17398
Kota, Parvathi; Reeves, Philip J; Rajbhandary, Uttam L et al. (2006) Opsin is present as dimers in COS1 cells: identification of amino acids at the dimeric interface. Proc Natl Acad Sci U S A 103:3054-9
Kim, Jong-Myoung; Hwa, John; Garriga, Pere et al. (2005) Light-driven activation of beta 2-adrenergic receptor signaling by a chimeric rhodopsin containing the beta 2-adrenergic receptor cytoplasmic loops. Biochemistry 44:2284-92
Eilers, Markus; Ying, Weiwen; Reeves, Philip J et al. (2002) Magic angle spinning nuclear magnetic resonance of isotopically labeled rhodopsin. Methods Enzymol 343:212-22
Klein-Seetharaman, J; Reeves, P J; Loewen, M C et al. (2002) Solution NMR spectroscopy of [alpha -15N]lysine-labeled rhodopsin: The single peak observed in both conventional and TROSY-type HSQC spectra is ascribed to Lys-339 in the carboxyl-terminal peptide sequence. Proc Natl Acad Sci U S A 99:3452-7
Reeves, Philip J; Kim, Jong-Myoung; Khorana, H Gobind (2002) Structure and function in rhodopsin: a tetracycline-inducible system in stable mammalian cell lines for high-level expression of opsin mutants. Proc Natl Acad Sci U S A 99:13413-8
Niu, Li; Kim, Jong-Myoung; Khorana, H Gobind (2002) Structure and function in rhodopsin: asymmetric reconstitution of rhodopsin in liposomes. Proc Natl Acad Sci U S A 99:13409-12
Reeves, Philip J; Callewaert, Nico; Contreras, Roland et al. (2002) Structure and function in rhodopsin: high-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N-acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line. Proc Natl Acad Sci U S A 99:13419-24
Altenbach, C; Cai, K; Klein-Seetharaman, J et al. (2001) Structure and function in rhodopsin: mapping light-dependent changes in distance between residue 65 in helix TM1 and residues in the sequence 306-319 at the cytoplasmic end of helix TM7 and in helix H8. Biochemistry 40:15483-92
Loewen, M C; Klein-Seetharaman, J; Getmanova, E V et al. (2001) Solution 19F nuclear Overhauser effects in structural studies of the cytoplasmic domain of mammalian rhodopsin. Proc Natl Acad Sci U S A 98:4888-92

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