Lightindependent signaling of different forms of the apoprotein

3.8.1 Opsin

In addition to activation of Gt by light-activated rhodopsin there is, in the absence of any chromophore, light-independent activation of Gt by the apoprotein opsin. In vitro, the rate of Gt activation by opsin is in the order of 10~6 of Mil at neutral pH [38]. Higher activities of opsin found in Gt activation assays eventually depend on residual retinal derivatives in the preparations [231]. Opsin appears also to exist in two conformational states [232], as is understood for other ligand-free GPCRs [233]. At neutral pH, opsin is in the inactive conformation which is stabilized by a salt bridge between Lys296 and Glu113 [39,120], a mechanism also used to stabilize the inactive rhodopsin ground state. Consequently, opsin mutants lacking the charge at either Lys296 or GluJ13 show enhanced basal activity, denoted constitutive activity [39]. As concluded from FTIR investigations, at low pH (pH < 5, depending on type and concentration of stabilizing anions) breaking of the salt bridge due to protonation of Glu"3 and concomitant conformational changes occur [232], This new conformation is similar to the Mil conformation, capable of binding C-terminal peptides of Gta, and represents the active conformation of opsin. The measured opsin activity would be high enough to have a physiological role in maintaining a certain stimulation of the visual cascade, which is one of the potential explanations for "bleaching desensitization" [234]. The desensitizing activity of opsin can be distinguished from the activity expressed in "photonlike noise" which was assigned to Mil formed via reversal of phosphorylation and arrestin binding [235],

3.8.2 Retinal-op sin complexes

The addition of all-Zra/w-retinal to opsin enhances its activity by the formation of non-covalent complexes [120,222,236], At a physiologically relevant 1:1

molar ratio, these ligand-like receptor*agonist complexes between opsin and all-?ra«s-retinal can activate Gt in the order of 0.5 (at pH 6.5) as well as Mil [237]. These non-covalent complexes can generate a conformation capable of interacting with Gt [120,236] and thus adopt a light-independent signaling state [236], This state can also interact with RK and arrestin [222,236,238],

Besides these non-covalent complexes, a second type of opsin*all-/ra«s-retinal complex exists. Schiff base formation does occur between Q.\\-trans-retinal and opsin, but not with the original Lys296 [236], leading to reversible "pseudo-photoproducts" [239] which interact with arrestin and kinase, but no interaction with Gt has been measured [239]. We may summarize the results as follows:

(A) all-/nms-retinal + opsin ^ opsin*all-fra«s-retinal

—> interaction with Gt, RK, arrestin

(B) all-ira^s-retinal + opsin ^ "pseudo-MIF"

—> interaction with RK, arrestin

Starting from both products, 11-ds-retinal can regenerate rhodopsin by binding to Lys296. Moreover, the all-iraws-retinal present does not compete with 11-a's-retinal, suggesting that in these products all-/ra«s-retinal occupies a different binding site [236]. The level of Gt activation of opsin^all-irafts-retinal complexes is strongly reduced when the palmitoyl groups are removed from Cys322 and Cys323 [237]. However, the removal of the palmitoyl anchors does not affect Mil activity. This suggests that the activity of opsin*all-ira«s-retinal complexes indeed arises not from small amounts of reversibly formed Mil, but from a separate form of active receptor with low intrinsic activity. Such active complexes may arise in vivo after the spontaneous decay of Mil by hydrolysis of the Schiff base. Binding of 11-as-retinal to opsin regenerates rhodopsin and provides a shut-off mechanism. During continuous illumination of the retina, opsin^all-irans-retinal may accumulate and play a role in the physiologic phenomenon of "bleaching desensitization".

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