Lipid requirements for rhodopsin regenerability

Hong, Keelung; Hubbell, Wayne L.

doi:10.1021/bi00746a033

Cited by 254 publications

(129 citation statements)

References 25 publications

(20 reference statements)

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“…ROS were isolated as described (11), except that the 35% (wt/vol) sucrose flotation step was carried out only once to minimize losses of the phosphodiesterase and transducin. Reconstituted membranes containing hydroxylapatite-purified rhodopsin and phosphatidylcholine at a molar ratio of 1:125 were prepared by dialysis (13). These membranes were devoid of phosphodiesterase and transducin.…”

mentioning

confidence: 99%

Flow of information in the light-triggered cyclic nucleotide cascade of vision.

Fung¹,

Hurley²,

Stryer³

1981

Proc. Natl. Acad. Sci. U.S.A.

680

284

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mentioning

confidence: 99%

Flow of information in the light-triggered cyclic nucleotide cascade of vision.

Fung¹,

Hurley²,

Stryer³

1981

Proc. Natl. Acad. Sci. U.S.A.

680

284

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“…To determine the concentration of rhodopsin, UROS membranes were solubilized in 2% CHAPS in the dark with 20 mM hydroxylamine and the absorption spectrum measured before and after bleaching under bright fluorescent light for 10 min as described (20). After subtracting the rhodopsin spectrum (normalized at 700 nm) measured in the light from the spectrum measured in the dark, the concentration of rhodopsin was calculated using a molar extinction coefficient of 42,700 M Ϫ1 cm Ϫ1 at 498 nm (22). In Vitro Phosphorylation of Bovine Rhodopsin-UROS membranes containing bovine rhodopsin were used as a substrate for phosphorylation by FLAG-tagged GRK1 and GRK7.…”

mentioning

confidence: 99%

Phosphorylation of GRK1 and GRK7 by cAMP-dependent Protein Kinase Attenuates Their Enzymatic Activities

Horner¹,

Osawa²,

Schaller

et al. 2005

Journal of Biological Chemistry

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Phosphorylation of G protein-coupled receptors is a critical step in the rapid termination of G protein signaling. In rod cells of the vertebrate retina, phosphorylation of rhodopsin is mediated by GRK1. In cone cells, either GRK1, GRK7, or both, depending on the species, are speculated to initiate signal termination by phosphorylating the cone opsins. To compare the biochemical properties of GRK1 and GRK7, we measured the K m and V max of these kinases for ATP and rhodopsin, a model substrate. The results demonstrated that these kinases share similar kinetic properties. We also determined that cAMP-dependent protein kinase (PKA) phosphorylates GRK1 at Ser 21 and GRK7 at Ser 23 and Ser 36 in vitro. These sites are also phosphorylated when FLAGtagged GRK1 and GRK7 are expressed in HEK-293 cells treated with forskolin to stimulate the endogenous production of cAMP and activation of PKA. Rod outer segments isolated from bovine retina phosphorylated the FLAG-tagged GRKs in the presence of dibutyryl-cAMP, suggesting that GRK1 and GRK7 are physiologically relevant substrates. Although both GRKs also contain putative phosphorylation sites for PKC and Ca 2؉ /calmodulin-dependent protein kinase II, neither kinase phosphorylated GRK1 or GRK7. Phosphorylation of GRK1 and GRK7 by PKA reduces the ability of GRK1 and GRK7 to phosphorylate rhodopsin in vitro. Since exposure to light causes a decrease in cAMP levels in rod cells, we propose that phosphorylation of GRK1 and GRK7 by PKA occurs in the dark, when cAMP levels in photoreceptor cells are elevated, and represents a novel mechanism for regulating the activities of these kinases. G protein-coupled receptors (GPCRs)1 belong to the largest family of signal-transducing proteins in eukaryotes. They mediate cellular responses to a variety of environment stimuli, including light, taste, odorants, ions, nucleotides, peptides, and lipids, through the activation of heterotrimeric G proteins (1-4). Phosphorylation of ligand-activated GPCRs by G proteincoupled receptor kinases (GRKs) is the initial step in the rapid termination or desensitization of GPCR signal transduction. For example, in rod cells of the vertebrate retina, desensitization occurs when light-activated rhodopsin is phosphorylated by GRK1, followed by the binding of visual arrestin to the phosphorylated rhodopsin, which blocks its interaction with transducin, the rod cell G protein (5). Although desensitization also occurs in cone cells, less is known about the proteins involved and their regulation. GRK1 and a cone-specific GRK, GRK7, are coexpressed in human and monkey cone cells (6 -8) where both may contribute to the deactivation of cone opsins (7-9). In contrast, only GRK7 is expressed in the cones of pigs and dogs. However, it is absent from the mouse genome altogether and mouse cones express only GRK1 (7). Therefore, differences in cone visual transduction between species may result in part from variations in the expression pattern of GRK1 and GRK7. We recently implicated GRK7 in the phosphorylation of cone o...

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“…Most significant is that regeneration of the absorption spectrum of rhodopsin in compressed films occurs without the addition of cis retinal, phospholipid or isomerase. This is in sharp contrast with regeneration studies of rhodopsin in solution where the addition of 11-cis retinal or isomerase is required [3,6].…”

Section: Resultsmentioning

confidence: 93%

“…It has been reported [3,5,6] that phospho lipid is necessary to regenerate rhodopsin. Hubbell [4] proposed that the regeneration of bleached rhodopsin may depend on the protein environment rather than phospholipid content.…”

Section: ]mentioning

confidence: 99%

Stability and Regeneration of Rhodopsin Absorption Spectra at an Air-Water Interface

Karan

Hirsch

Brody

1978

Zeitschrift Für Naturforschung C

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Vision, Rhodopsin, Monolayers, Membranes, Photobiology Stability and regenerability of the absorption spectrum of CTAB solubilized rhodopsin at an air-water interface is studied. The spectral properties of rhodopsin films in the dark are stable more than 150 minutes. When rhodopsin is bleached (40 -50%) and maintained in highly com pressed films recovery of the spectrum is observed. The recovery is 57% to 100% of the pigment present before irradiation. At low surface pressure or if the films are expanded after irradiation there is no observable recovery.

show abstract

Lipid requirements for rhodopsin regenerability

Cited by 254 publications

References 25 publications

Flow of information in the light-triggered cyclic nucleotide cascade of vision.

Flow of information in the light-triggered cyclic nucleotide cascade of vision.

Phosphorylation of GRK1 and GRK7 by cAMP-dependent Protein Kinase Attenuates Their Enzymatic Activities

Stability and Regeneration of Rhodopsin Absorption Spectra at an Air-Water Interface

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