Arrestin-Rhodopsin Binding Stoichiometry in Isolated Rod Outer Segment Membranes Depends on the Percentage of Activated Receptors

Abstract

In the rod cell of the retina, arrestin is responsible for blocking signaling of the G-protein-coupled receptor rhodopsin. The general visual signal transduction model implies that arrestin must be able to interact with a single light-activated, phosphorylated rhodopsin molecule (Rho*P), as would be generated at physiologically relevant low light levels. However, the elongated bi-lobed structure of arrestin suggests that it might be able to accommodate two rhodopsin molecules. In this study, we directly addressed the question of binding stoichiometry by quantifying arrestin binding to Rho*P in isolated rod outer segment membranes. We manipulated the "photoactivation density," i.e. the percentage of active receptors in the membrane, with the use of a light flash or by partially regenerating membranes containing phosphorylated opsin with 11-cis-retinal. Curiously, we found that the apparent arrestinRho*P binding stoichiometry was linearly dependent on the photoactivation density, with one-to-one binding at low photoactivation density and one-to-two binding at high photoactivation density. We also observed that, irrespective of the photoactivation density, a single arrestin molecule was able to stabilize the active metarhodopsin II conformation of only a single Rho*P. We hypothesize that, although arrestin requires at least a single Rho*P to bind the membrane, a single arrestin can actually interact with a pair of receptors. The ability of arrestin to interact with heterogeneous receptor pairs composed of two different photo-intermediate states would be well suited to the rod cell, which functions at low light intensity but is routinely exposed to several orders of magnitude more light.The hundreds of different types of G-protein-coupled receptors (GPCRs) 2 and their binding partners represent versatile protein families, whose individual members have been modified by nature to accomplish many different functions while preserving a basic structure and activation mechanism (1, 2). GPCRs share a common seven-transmembrane helical structure, which, when activated by ligand or stimuli, binds and activates G-proteins to initiate cell signaling (3). Most GPCRs also share a common mechanism of signal termination, which involves receptor phosphorylation and binding of the protein arrestin. However, the fates of arrestin-bound receptors vary widely depending on the type of receptor and its bound ligand. Arrestin-bound receptors may be internalized and degraded, internalized and recycled, or even initiate Gprotein independent signaling (4).Although detailed structural information on different GPCRs (5-9) and arrestins (10 -13) has been available for some time, basic questions regarding their interaction remain unanswered. In particular, the binding stoichiometry (how many active receptors does a single arrestin bind?) is a matter of debate. Historically, one-to-one binding has always been assumed, because many GPCRs are present at low concentrations on the cell surface (1), or in the case of the photoreceptor rhodopsin, activ...

Conformation of receptor-bound visual arrestin

“…S6 and SI Materials and Methods). Therefore, we conclude that the DEER measurements apply to a high-affinity one-to-one arrestin- P-Rh* complex, consistent with the finding that regardless of the binding stoichiometry, arrestin-1 stabilizes the active state (metarhodopsin II) of only one rhodopsin (42). However, the data do not eliminate the possibility of complexes with other stoichiometry in the native disk membrane that do not alter arrestin conformation.…”

“…In this case, a significant domain movement does not seem to be necessary. On the other hand, it may be that the N domain and finger-loop region are the primary sites of interaction with P-Rh* and that the C domain has an accessory function, including the possibility of interaction with another receptor molecule in an inactive state (42,43), the membrane surface, or other signaling proteins. Among the four vertebrate subtypes, arrestin-1 shows the highest receptor specificity and selectivity for P-Rh* (50).…”

Functional map of arrestin-1 at single amino acid resolution

“…Principally, the reorganized interface could thus provide a secondary binding site for the accommodation of a GPCR dimer. Indeed it has been suggested that the arrestin C loop binds a second rhodopsin molecule in disc membranes containing high levels of activated rhodopsin (18,42). Site-directed spin-labeling studies, on the other hand, revealed a high flexibility of this arrestin region upon binding to both highly activated P-ROS* and nanodiscs containing only monomeric P-rhodopsin* (28).…”

Involvement of distinct arrestin-1 elements in binding to different functional forms of rhodopsin

“…Monomeric rhodopsin was shown to be necessary and sufficient for high-affinity arrestin-1 binding (26,45). However, recent reports suggest that two types of arrestin-1-rhodopsin complexes are formed upon activation of a high fraction of rhodopsin in native disk membranes, with 1:1 and 1:2 arrestin: rhodopsin stoichiometries (46,47). In the latter, arrestin-1 apparently binds only one rhodopsin with high affinity, stabilizing its Meta II state (46) and trapping all-transretinal (47), whereas the other molecule is engaged with much lower affinity by distinct arrestin-1 elements localized in the C-domain (47).…”

“…However, recent reports suggest that two types of arrestin-1-rhodopsin complexes are formed upon activation of a high fraction of rhodopsin in native disk membranes, with 1:1 and 1:2 arrestin: rhodopsin stoichiometries (46,47). In the latter, arrestin-1 apparently binds only one rhodopsin with high affinity, stabilizing its Meta II state (46) and trapping all-transretinal (47), whereas the other molecule is engaged with much lower affinity by distinct arrestin-1 elements localized in the C-domain (47). All forms of rhodopsin used here were solubilized by a large excess of bicelles and most of the perturbations were observed to be in the N domain of arrestin-1 and in the C terminus that is anchored to this part of the molecule (Figs.…”