Investigation of the organization of rhodopsin in the sheep photoreceptor membrane by using cross-linking reagents

Brett, M.; Findlay, John B. C.

doi:10.1042/bj1770215

Cited by 32 publications

(16 citation statements)

References 30 publications

(26 reference statements)

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“…A characteristic distribution of oligomers (n-mers) was observed in which the yield of oligomer decreased as n increased. This pattern has been observed in sheep photoreceptor membranes (31) and was interpreted to be indicative of monomeric rhodopsin. Extension of the glutaraldehyde cross-linking studies reported here ( 19) indicates that dark-adapted rhodopsin is indeed monomeric and that dichroic ratio somewhat <3.…”

Section: Cross-linking Of Rhodopsin In Photoreceptor Membranessupporting

confidence: 66%

Transient dichroism in photoreceptor membranes indicates that stable oligomers of rhodopsin do not form during excitation

Downer¹,

Cone²

1985

Biophysical Journal

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If a photoexcited rhodopsin molecule initiates the formation of rhodopsin oligomers during the process of visual excitation, the rate of rotational diffusion of the rhodopsin molecules involved should change markedly. Using microsecond-flash photometry, we have observed the rotational diffusion of rhodopsin throughout the time period of visual excitation and found that no detectable change occurs in its rotational diffusion rate. Partial chemical cross-linking of the retina yields oligomers of rhodopsin and causes a significant decrease in the rotational diffusion rate of rhodopsin even when as little as 20% of rhodopsin is dimeric. Moreover, the pattern of oligomers formed by cross-linking, taken together with the magnitude of decreases in rotational diffusion rate accompanying the cross-linking reaction, suggests that rhodopsin is a monomer in the dark-adapted state. The experiments reported here show that photoexcited rhodopsin molecules do not irreversibly associate with unbleached neighbors during the time course of the receptor response. Hence, it is not likely that stable oligomers of rhodopsin trigger the excitation of the photoreceptor cell.

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Section: Cross-linking Of Rhodopsin In Photoreceptor Membranessupporting

confidence: 66%

Transient dichroism in photoreceptor membranes indicates that stable oligomers of rhodopsin do not form during excitation

Downer¹,

Cone²

1985

Biophysical Journal

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“…Additionally, biophysical measurements using high speed flash photometry and microspectrophotometry have shown that Rho undergoes rapid rotational and lateral diffusion (9, 10). Cross-linking studies have also suggested a monomeric organization for rhodopsin (11,12). Accordingly, the concept of how Rho functions in the disk membrane of retinal ROS has been dominated by the hypothesis that Rho rapidly diffuses as a monomeric unit in the fluid membranes, which are mostly composed of highly unsaturated…”

mentioning

confidence: 99%

The Hydrodynamic Properties of Dark- and Light-activated States of n-Dodecyl β-D-Maltoside-solubilized Bovine Rhodopsin Support the Dimeric Structure of Both Conformations

Medina

Perdomo

Bubis

2004

Journal of Biological Chemistry

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Rhodopsin (Rho) has been extracted in n-dodecyl ␤-Dmaltoside (DM) from bovine retinal rod outer segments and purified to homogeneity by affinity chromatography on concanavalin A-Sepharose. Because chemical cross-linking of Rho and photoactivated Rho (Rho*) provided initial evidence for the oligomeric nature of the photoreceptor protein, we carried out a hydrodynamic characterization of the native and activated conformations of detergent-solubilized Rho. The molecular weights of the complexes between dark and photoexcited states of Rho and DM were determined by gel filtration chromatography on Sephacryl S-300, in the presence of 0.1% DM. Subtracting the size of the corresponding detergent micelles resulted in molecular masses of 78 kDa for native Rho and 76 kDa for Rho*. The measured content of 0.97 g of detergent/g of protein resulted in a calculated partial specific volume of 0.765 cm 3 /g for the protein-detergent complex and a molar mass of 64 -65 kDa for the protein moiety. The sizes of Rho⅐DM and Rho*⅐DM complexes were also evaluated by sedimentation on 10 -30% sucrose gradients, in the presence of 0.1% DM, and molecular masses of about 60 kDa were estimated for both the dark-and light-activated states of the photoreceptor protein. The size of Rho was determined to be 65,300 and 69,800 Da, respectively, when the purified Rho⅐DM complex was either chromatographed on Sephacryl S-300 or ultracentrifuged on sucrose gradients in the absence of DM. All these results were consistent with a dimeric quaternary structure for both conformations of Rho. Additionally, the functional integrity of the purified photoreceptor protein following gel filtration chromatography and ultracentrifugation was demonstrated by three criteria as follows: (i) its characteristic UV-visible absorption spectra, (ii) its capability to photoactivate transducin, and (iii) its ability to serve as a substrate for rhodopsin kinase.G protein-coupled receptors (GPCRs) 1 are a large group of integral membrane proteins that respond to environmental signals and initiate transduction pathways that activate cellular processes. In general, the activation of the receptor by binding of an extracellular signal or by light absorption triggers a conformational change in its structure, which then activates a peripherally membrane-associated heterotrimeric G protein. One of the most important unanswered questions is how these receptors operate and couple to their cognate G proteins. A growing body of recent pharmacological, biochemical, and biophysical data strongly suggests that GPCRs are organized as functional homo-and heterodimers as well as higher order oligomers (1, 2). Oligomerization of GPCRs may cluster these receptors in particular regions of the membrane. This process could be critical for the proper kinetics of GPCR signaling, selectivity, desensitization, and internalization. Receptor maturation during biosynthesis and translocation to the plasma membrane could also benefit from oligomerization (3). Additionally, the formation of heteromers may ex...

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“…Purified photoreceptor discs from porcine and equine retinas were prepared as described by Brett & Findlay (1979); no attempt was made to label the proteins with [3H]retinal and all procedures were performed in daylight at 4°C. The discs were digested with V8, pelleted and the V8-S fragments prepared as described (Findlay et al, 1981.…”

mentioning

confidence: 99%

“…Crude ROS membranes were prepared from ovine retinas by the method of Brett & Findlay (1979), with 67mM-sodium phosphate, pH7.0, instead of the 0.1M-Tris/HCl buffer previously employed. Rhodopsin content was calculated from the absorbance changes at 500nm (AA500) before and after photobleaching of solutions in 2% (w/v) Emulphogene BC 720 containing 50mM-neutral NH2OH (adjusted to pH7.0 with NaOH).…”

mentioning

confidence: 99%

Sequence variability in the retinal-attachment domain of mammalian rhodopsins

Pappin¹,

Findlay²

1984

Biochemical Journal

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Investigation of the organization of rhodopsin in the sheep photoreceptor membrane by using cross-linking reagents

Cited by 32 publications

References 30 publications

Transient dichroism in photoreceptor membranes indicates that stable oligomers of rhodopsin do not form during excitation

Transient dichroism in photoreceptor membranes indicates that stable oligomers of rhodopsin do not form during excitation

The Hydrodynamic Properties of Dark- and Light-activated States of n-Dodecyl β-D-Maltoside-solubilized Bovine Rhodopsin Support the Dimeric Structure of Both Conformations

Sequence variability in the retinal-attachment domain of mammalian rhodopsins

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