2.5.1 Activation of phototransduction
In view of the prototypical structure of a GPCR, there is no doubt that in phototransduction the transition of rhodopsin into an active metarhodopsin state is transmitted to downstream components of the phototransduction cascade via interaction with a heterotrimeric G-protein. Retinal-binding proteins which are distantly related to rhodopsin, for example the retinochromes of cephalopods, show an overall topology that is similar to that of rhodopsins. Retinochromes, however, act as photoisomerases. They are integral members of a shuttle system which provides retinal in its 11 -cis configuration for rhodopsin synthesis and regeneration [116,117]. The non-transducing function of retinochromes is clearly mirrored in the absence of domains for G-protein interaction in the cytoplasmic loops i2 and i3 of this protein .
The current state of research suggests that intercalary evolution of rhodop-sin-G-protein coupling in invertebrates has assembled at least two distinct types of phototransduction cascades. The first pathway operates in depolarizing, microvillar (rhabdomeral) photoreceptors, the second in hyperpolarizing, ciliary photoreceptors. In microvillar photoreceptors, rhodopsin couples upon light activation to the Gq subtype known to activate phospholipase C (PLC(3) as effector enzyme. The sequential interaction of rhodopsin, Gq and PLC has been firmly established for microvillar photoreceptors of cephalopods, crustacea and insects [118-134], The genes encoding the three subunits (dgq, dbe, dge) of the visual Gq-protein of fly photoreceptors [127,135-137] and cephalopod photoreceptors [138-141] have been cloned and sequenced. A sequence comparison between the visual G-protein subunits of Drosophila and the subunits of other G-proteins identifies the visual G-protein as Gq subtype [127,137], The primary structure of GqP bears no function related sequence conservation , The amino acid sequence of Gqy, however, reveals a distant relationship to gamma-subunits of vertebrate transducins. Thus, rhodop-sin and Gqy are members of phototransduction pathways that are conserved irrespective of the photoreceptor cell type and the effector enzyme activated. Drosophila mutants in the genes coding for the Gqa and GqP subunits demonstrate that Gqa is required for the activation of phototransduction while Gq(3 is essential for the interaction of Gqp with metarhodopsin . The crucial role of phospholipase C in phototransduction was demonstrated most clearly in Drosophila by isolating the norpA gene, which encodes for a phospholipid-specific phosphodiesterase abundantly expressed in the retina [129,130]. Strong alleles of norpA completely abolish the light response [143,144].
Thus, up to the point of PLC activation, the initial stages of the visual cascade in microvillar photoreceptors appear to be rather similar. There is, however, no final answer to the essential question whether the transduction mechanisms diverge among invertebrates at later stages of the phototransduction cascade. PLC activated by Gq is known to catalyse the hydrolysis of the membrane lipid phosphatidyl inositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) [13,18], Both IP3and DAG have been implicated in the light-initiated opening of cation channels. There is evidence indicating that the opening of cation channels in the ventral nerve photoreceptor of Limulus involves an IP3-mediated calcium release from lP3-sensitve calcium stores , The expression of a cyclic GMP gated channel subunit in these cells raises the possibility that cGMP is involved in phototransduction , In Drosophila, the opening of the light-activated channel has been attributed to the messenger functions of DAG and DAG metabolites, in particular of polyunsaturated fatty acids (PUFAs) [147,148], while PUFAs appear not to activate phototransduction in Limulus , The ion channels activated in response to a light stimulus have been unequivocally identified in fly photoreceptors - they are members of the TRP protein family [149-154],
Discussion of the general design and the functional role of individual stages downstream of Gq activation recently took a new turn after it had been shown that in fly photoreceptors the proteins involved in phototransduction are assembled into a supramolecular signalling complex (Figure 8). This complex is organized by the scaffold protein INAD (inactivation no afterpotential) via the binding of protein ligands to its PDZ domains [155-158]. The functional role of the proteins assembled in this complex is indicated by the phototransduction defects caused by mutations of the respective genes. The ligands make up the norpA (no receptor potential A) encoded PLC, the inaC (inactivation no afterpotential) encoded eye-specific protein kinase C (ePKC) and the trp (transient receptor potential) encoded major light-activated cation channel
Figure 8. Model for the molecular design of the phototransduction machinery in Drosophila photoreceptor cells. A light-activated rhodopsin (R*, left) interacts with the heterotrimeric G-protein, Gq, which results in the dissociation of Gq into Gqc(-GTP and GqPy (right). The activated Gqo( serves as a molecular shuttle which transmits activation of the visual pigment to the target enzyme PLCp. PLC(3 is anchored via the PDZ-domain protein INAD to the so-called INAD signalling complex, which further contains an eye-specific protein kinase C (ePKC) and the ion channel TRP. Through the unconventional myosin NINAC, the signalling complex is anchored to the actin cytoskeleton. One core complex, forming an intact TRP ion channel, is composed of four TRP molecules and the corresponding numbers of INAD, ePKC and PLC. The second messengers, IP3 and diacylglyerol, generated by the action of PLCP, are thought to activate and modulate the influx of Ca2+ ions through the TRP channels. Model modified from  and ; see these references for an in-depth discussion.
TRP. INAD has been shown to interact via homomeric interactions with other INAD molecules. In this way the core proteins associated by INAD may become organized in a more extended web . Further transduction-relevant proteins reported to bind to INAD are the unconventional myosin (NINAC, neither inactivation nor afterpotential), the second light-activated channel protein (TRPL, trp-like) and even rhodopsin .
Figure 8 summarizes some important features of the structure and function of the INAD signalling complex. INAD, PLC, ePKC and TRP can be reliably isolated together by co-immunoprecipitation experiments in a rather invariable 1:1:1:1 stoichiometry . The pattern of their binding to the five PDZ domains of INAD as summarized by Huber  has been evaluated in detail. That these proteins constitute a functional unit is clearly demonstrated by the finding that ePKC present in the isolated complex catalyses the DAG- and calcium-dependent phosphorylation of TRP as well as of INAD [155,160]. They are, therefore, regarded as the core complex of a larger transduction unit (transducisome)  which would include about 15-25 TRP channels in tetrameric form. In addition, the complex might be linked to actin filaments located in the microvillar lumen via the binding of the unconventional myosin NINAC to INAD. Direct proof for this interaction has, however, not yet been presented. Calmodulin (CaM), which has been shown to interact with the various CaM binding sites present on the members of the complex , is omitted from the scheme. In Figure 8 it is also emphasized that rhodopsin is present in the membrane in a large excess to INAD signalling complexes. Estimates on the basis of a homomeric TRP channel composition suggest that a single microvillus may contain only about 25 INAD signalling complexes . The shuttle function for the information transfer from photon capture by an individual rhodopsin molecule to INAD-linked PLC is attributed to Gqa. Light-dependent as well as light-independent activation of Gq reveals that Gqa but not Gqpy interacts with INAD-linked PLC , If the hydrolysis of GTP is prevented by replacing GTP with GTP-y-S, the INAD-linked PLC molecules form a stable high-affinity complex with Gqa . There is, however, no evidence that under these conditions (activated) rhodopsin is also complexed with INAD. The effect of the activation of INAD-linked PLC on the inositol phospholipid composition in the vicinity of the complex has not yet been explored. The activated membrane patch in Figure 8 indicates that activation of PLC and the localized ion influx through INAD-linked TRP channels are likely to create transient inhomogeneities in the inositol lipid derived messengers as well as in Ca2+, which would have consequences for excitation as well as termination of the visual response ,
Some of the implications of assembling key members of the phototrans-duction cascade into a supramolecular complex are: the members of the core complex become correctly targeted in a defined stoichiometry to the microvil-lar photoreceptor membrane and remain retained in this compartment . Furthermore, the close proximity of proteins involved in the generation and control of the visual response appears to ensure signal amplification, high specificity and sensitivity of signalling as well as high speed of signalling, i.e. short latencies for activation and termination of light responses [9,18,157], It has been concluded that a single complex (transducisome) represents the functional unit for the generation of a quantum bump, the response of which is elicited by absorption of a single photon at a rhodopsin molecule , Whether the organization principle of phototransduction in fly photoreceptors also holds for other microvillar photoreceptor systems is not yet known. It is to be expected that signalling complexes constitute the basis for high speed signalling primarily in the photoreceptors of insects. Thus, in addition to modifications in the type of second messenger there may be another point of divergence in invertebrate phototransduction mechanisms, which concerns the organization of signalling cascade components in heteromultimeric protein complexes.
Invertebrates have developed at least one rhodopsin-activated phototransduction pathway which does not involve the activation of PLC. Such a pathway is realized in hyperpolarizing ciliary photoreceptors of molluscs, such as the scallop. Cumulative evidence suggests that light-activated rhodopsin here couples to a Go subtype  which in turn activates guanylate cyclase as effector enzyme . In fact hyperpolarization of these ciliary photoreceptors has been shown not to result from the blocking of cGMP gated ion channels as in vertebrate photoreceptors but from the opening K+ selective ion channel by cyclic GMP . Thus, as in vertebrate photoreceptors, the light-activated conductance is controlled by cyclic GMP (see Chapter 3), but the activation parameters for the key enzymes involved in the control of cyclic GMP levels, cGMP phosphodiesterase and guanylate cyclase, have been reverted.
Equally important as the activation of phototransduction is the efficient inactivation of each step of the transduction cascade. Only highly effective inactivation mechanisms enable a transduction cascade to repeatedly transmit information with high temporal fidelity. The necessity for an effective deactivation of the active rhodopsin state is particularly evident in invertebrate photoreceptors in which light-absorption triggers the formation of a long-lived active metarhodopsin state , Most of the information on deactivation of active metarhodopsin comes from studies on dipteran flies (Drosophila, Calliphora), from which two genes encoding visual system-specifically expressed arrestins, arrestin 1 (Arrl) and arrestin 2 (Arr2), have been isolated [170-173]. Analysis of arrl- and arr2-mutants in Drosophila showed that both arrestin isoforms contribute to the termination of the phototransduction cascade . Arr2, the major arrestin form present in the photoreceptor cell, binds light-dependently with high affinity to metarhodopsin (Figure 9) [73,95,96,175]. This interaction constitutes the rate-limiting step in the overall termination of the light response . Wild-type Drosophila contain more rhodopsin molecules than Arr2 molecules. Therefore, irradiation of Rhl-containing photoreceptors with blue light, which shifts about 70% of Rhl rhodopsin into metarhodopsin (Figure 9), creates a large excess of activated metarhodopsin over Arr2. Under these circumstances, i.e. in a situation unlikely to occur under normal light conditions, the photoreceptor cell generates a sustained electrical response. This prolonged depolarizing afterpotential (PDA) terminates very slowly after the cessation of light, unless metarhodopsin is photoconverted back into rhodopsin [64,176-179] (Figure 9). The conversion of about 20% of Rhl rhodopsin present in the fly photoreceptor into metarhodopsin is sufficient to elicit a PDA. It is thus concluded that the molar ratio of Rhl to Arr2 within a photoreceptor cell is around 5 : 1 . In flies, the PDA may last from minutes to hours, which indicates that the stable M-state itself and not a transient intermediate has the ability to excite the photoreceptor for a rather long time.
Both arrestins are phosphorylated light-dependently by a Ca2+/calmodulin-dependent kinase (CaM Kinase) [173,180-182]. For Arr2, this phosphorylation was shown to be a prerequisite for its release from the visual pigment after photoreconversion of M into P , But, as indicated in Figure 5, in a distinct difference to the arrestin-mediated deactivation of vertebrate rhodopsin, phosphorylation of metarhodopsin in flies is not a prerequisite for binding of Arr2 [8,95,98]. It seems rather that binding of Arr2, similar to P-arrestin binding to P-adrenergic receptors [183-185], is part of a recruitment mechanism for rhodopsin internalization allow metarhodopsin to become phosphorylated by a rhodopsin kinase  and preventing metarhodopsin dephosphorylation by the receptor phosphatase RDGC [96,98,186] (see also Section 2.3.2). In a second distinct difference to the situation in vertebrate photoreceptors, phosphorylation of metarhodopsin, at least in flies, is not linked to receptor inactivation . Since visual arrestins have also been isolated from
I blue light
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