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

Abstract

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.

A Rhodopsin Mutant Linked to Autosomal Dominant Retinitis Pigmentosa Is Prone to Aggregate and Interacts with the Ubiquitin Proteasome System

“…aggregation, and not inclusion body formation, that underlies FALS and perhaps other aggregation-linked neurodegenerative diseases. The oligomeric forms of rhodopsin, much like those of SOD, are non-native structures, given the fact that the wild-type is monomeric (33). Thus, they fit our definition of aggregates, being "non-native oligomers.…”

“…Aggresome formation by P23H does not require proteasome inhibitors, because they are observed in cells in the absence of any drug treatment. Second, because FRET is efficient only for C/YFP fluorophores that are within 100 Å of each other (54), our data suggest that the P23H mutation endows the rhodopsin molecule, normally a monomer even at the high concentrations present within the rod outer segments (33), with the ability to self-associate into compact structures. Finally, we observe identical patterns of electrophoretic motilities of P23H rhodopsin when the cells are lysed in the presence of alkylating agents such as iodoacetamide, which prevent the formation of non-native disulfide-linked species in the highly oxidizing environment of SDS-PAGE (55).…”

“…The majority of P23H, however, migrated as dimers and higher molecular weight oligomers. These oligomers are evidence of a non-native conformation, because rhodopsin is monomeric in its native membrane (33). A substantial fraction of P23H partitioned into the detergent-insoluble fraction, suggesting that P23H is more prone to aggregate than wild-type rhodopsin (Fig.…”

Trafficking of G Protein–Coupled Receptors

“…Early studies using rhodopsin, 17,18 muscarinic, 19 and ␤-adrenergic 20 receptors as model GPCRs suggested that GPCRs exist primarily as monomers, although modification of the detergent extraction systems used for protein purification led early investigators to suggest that a varying fraction of GPCRs may also be present in oligomeric form. 19,20 Much recent work using coimmunoprecipitation and resonance energy-transfer techniques have convincingly demonstrated that dimeric GPCR structures are present at the plasma membrane and are the topic of several recent reviews.…”

The supramolecular structure of the GPCR rhodopsin in solution and native disc membranes

“…In most of these studies, photoactivation of rhodopsin did not affect the results (see also ref. [56]), suggesting that this lack of pre-organization did not change upon activation of rhodopsin.…”