Key motifs of invertebrate rhodopsins

A detailed structure comparison of invertebrate rhodopsins has recently been performed by Gärtner [14]. To highlight some of the key motifs for rhodopsin function in the following chapters of this review, a coding sequence comparison is carried out for Drosophila melanogaster. This is the only insect species in which six out of seven expressed opsins genes (rhl to rh6) have not only been sequenced, but the corresponding rhodopsins have been functionally characterized as well [35^-1]. The Drosophila rhodopsins compared in the alignment of Figure 4 are expressed in the compound eye (Rhl, Rh3 to Rh6) and in extra-compound eye structures such as the adult ocellar photoreceptors and the testis (Rh2) as well as in larval photoreceptors (Rhl, Rh3, Rh4) [42], As mentioned above, Rh7 identified by the Drosophila genome project [32] is still in an orphan state due to the lack of data for the expression pattern of the gene and the function of the protein. In the alignment depicted in Figure 4, the 7TM-structure of the Drosophila rhodopsins is founded on hydrophobicity calculations [14]. Experimental studies exploring the transmembrane location of the 7 transmembrane helices of distinct Drosophila rhodopsins in more detail have not yet been carried out.

2.3.1 Positionally conserved domains

The positions of domains conserved in Rhl to Rh6 of Drosophila are summarized in the structure model shown in Figure 4. These domains are of particular interest since the light-activated state of each rhodopsin activates the same visual G-protein (Gq) with the same efficiency, regardless of the structural differences determining the particular absorption properties of a rhodopsin. This has been demonstrated most convincingly by the functional expression of rhodopsins Rh2 to Rh6 in photoreceptor cells Rl-6 in place of the intrinsic


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Figure 4. Conserved amino acids and functional domains in Drosophila rhodopsins. Structure model of Drosophila Rhl rhodopsin, amino acids are shown in one letter code. The C-termini and N-termini of Rh2 to Rh6 are aligned with those of Rhl. Conserved amino acids and functional domains are highlighted in the figure and explained in the text.

Rhl rhodopsin [43]. Thus, these rhodopsins share a common interface for the interaction with the G-protein (Gq) coupled to Rhl. As all of the ecto-pically expressed rhodopsins are properly targeted to the rhabdomeral photo-transduction compartment in Rl-6 photoreceptor cells, they have to provide a common interface for cellular targeting as well as a common interface for the activation and control of phototransduction. This conservation in the structure of rhodopsin extends beyond the rhodopsins expressed in Drosophila photoreceptors, as the heterologous expression of opsin genes encoding the UV- and blue-light absorbing rhodopsins of the honey bee {Apis mellifera) and of a locust rhodopsin {Schistocerca gregarina) fully rescues signal transduction in null-mutants of Drosophila Rhl [44,45].

As indicated in Figure 4, highly conserved amino acids in Drosophila rhodopsins are located in cytoplasmic loops il to i3 and in extracellular loop e2. A similar scheme of overlapping conserved sites is observed if rhodopsins from other insect species are included in the structure comparison, e.g. the rhodopsins cloned from the honey bee [45,46] or from the sphingid moth Manduca sexta [25]. In contrast to vertebrate rhodopsins (see Chapter 3), no unequivocal evidence is available on the role of conserved amino acids with regard to forming an interface with the visual G-protein and arrestin isoforms, which control the active rhodopsin state (metarhodopsin). The finding that mutations of highly conserved amino acids (L81Q, N86I) in the first cytoplasmic loop and in the extracellular loop e2 (E194K, G195) of Drosophila Rhl arrest rhodopsin synthesis in a nascent state suggests that this loop harbours information required in initial steps of the targeting process to the photoreceptor membrane [47]; for summaries see [48,49]. In addition to extended domains, amino acids are positionally conserved at single sites in all rhodopsins. This holds particularly for two cysteines (CI23 and C200 of Drosophila Rhl, Figure 4) which form a structure-stabilizing disulfide bridge on the extracellular surface of each rhodopsin molecule [14].

2.3.2 The chromophore binding site

Positional conservation in particular includes the chromophore binding site, a lysine residue in transmembrane helix VII (corresponding to K319 of Drosophila Rhl in Figure 4). In invertebrate rhodopsins, this site is occupied by a variety of retinaldehyde-related chromophores in the 11 -cis conformation (retinal, 3-hydroxyretinal, 4-hydroxyretinal) [50-53].

Raman resonance measurements in cepahalopod rhodopsin [54,55] and in an insect UV-rhodopsin [56], as well as infrared and cryogenic spectroscopy [57,58], indicate that the chromophore is covalently linked to the chromophore binding site via the protonated state of the Schiff base, as is the case in vertebrates. In vertebrates, the protonated Schiff base of rhodopsin is stabilized by a glutamic acid residue (El 13 of bovine rhodopsin) which serves as a negatively charged counter-ion to the positively charged chromophore [59-61], In most invertebrate rhodopsins, a conserved tyrosine residue (Y126 of Drosophila Rhl, see Figure 4) has been regarded as the prime candidate for the Schiff base counter-ion [14]. A denaturation-reprotonation study with cepha-lopod (Octopus) rhodopsin, however, does not support this view [62]. In the absence of other candidates for a corresponding counter-ion, it is thus proposed that Schiff base protonation of invertebrate rhodopsin does not require a stabilizing salt bridge with a negative charge [62].

Accordingly, a profound difference exists between vertebrate and invertebrate rhodopsins. This has to be seen in the context that cephalopod rhodopsin and most of the other insect rhodopsins investigated so far form, upon light absorption, a relatively stable metarhodopsin state [50,63-66], This state represents the active state of invertebrate rhodopsin, or at least a state spectrally closely related to the active state. From this state, in which the chromophore is still attached to opsin via a protonated Schiff base, the initial rhodopsin state is regained by photoregeneration via an intermediate with the chromophore in the W-cis configuration [63,64].

For Drosophila and related insects, the formation of a stable protonated Schiff base bond between the chromophore and opsin is a primary structural requirement for proper processing of newly synthesized rhodopsin and its transport to the rhabdomeral photoreceptor membrane. In ll-cw-3-hydroxyretinal-deficient flies, opsin synthesis is blocked at the post-translational level, in a nascent opsin state [67]. Opsin molecules which, due to the absence of an attached chromophore, cannot adopt the properly folded structure of a mature rhodopsin, are targeted into a degradation pathway. As a consequence, the opsin/rhodopsin density in the phototransducing rhabdomeral membrane compartment becomes drastically reduced [68], The requirement for a stabilized form of chromophore attachment extends beyond synthesis, targeting, activation and regeneration of rhodopsin. The renewal of rhodopsin involves selective internalization of metarhodopsin [69,70], and thus also relies on a stable metarhodopsin with a protonated Schiff base-bound chromophore.

In the visual cycle of vertebrates, Schiff base deprotonation and proton translocation to the counter-ion is part of the molecular mechanism of rhodopsin activation [71,72] (see Chapter 3). Relaxation of the opsin structure, which initiates Schiff base hydrolysis, the subsequent release of all-trans retinal from opsin and, finally, the formation of a new Schiff base bond between opsin and 11 -cis retinal are the hallmarks of the chemical regeneration of vertebrate rhodopsin (see Chapter 3). In invertebrates, it is questionable whether the Schiff base ever becomes deprotonated during a regular rhodopsin cycle. Basic steps of that cycle involve transitions which terminate at rhodopsin states with chromophore configurations possessing a stabilized, protonated Schiff base bond. How this stability is achieved is not yet known. Apart from unidentified intramolecular interactions, rhodopsin stability might be affected by intermolecular interactions. To this end it has been shown in situ that the interaction with arrestin significantly enhances the life-time of metarhodopsin. In vivo, the life-time of metarhodopsin is also extended in the absence of arrestin [73], which suggests the existence of additional stabilization mechanisms.

2.3.3 Three-dimensional structure

Despite the unique properties Drosophila offers for mutation analyses to solve structure-function relationships of invertebrate rhodopsin, advanced biophysical techniques are still limited to invertebrate species that provide sufficient rhodopsin for purification and crystallization. A recent study which sheds light on the three-dimensional structure of an invertebrate rhodopsin employed cryo-electronmicroscopy and image processing of 2D-crystals of squid rhodopsin [74]. Crystallization was achieved with a C-terminally truncated form of squid rhodopsin. The proteolytic truncation removed a proline-rich extension from the C-terminus which is unique for rhodopsins of cephalo-pods [75]. The projection structure of the crystallized squid rhodopsin at 8 A resolution revealed a high similarity in the rhodopsin topology to that of vertebrate rhodopsin at a comparable resolution [74,76,77]. Apart from some differences in the packing of the transmembrane helices and the presence of a well-ordered structure in loop i3, the fit of the 3D map of cephalopod rhodopsin with the 3D structure of bovine rhodopsin agrees with the evolutionary and structural conservation of rhodopsins from invertebrates to man. An interesting result derived from crystallization studies of squid rhodopsin concerns the existence of a linear lattice contact between rhodopsin molecules. This may be the structural basis for an ordered alignment of rhodopsins in the microvillar membranes of invertebrate rhabdomeral photoreceptors. The fixed orientation of invertebrate rhodopsin in such a lattice may account for the ability to analyse the plane of polarized light [74], The finding of a lateral lattice organization of squid rhodopsin may imply that the remaining members of the phototransduction cascade are also organized into superstructures [78]. In Drosophila, such a multimeric signalling complex is organized via the scaffolding-protein INAD (see Section 2.5.1).

2.3.4 Post-translational modification: palmitoylation, glycosylation and phosphorylation

The excellent fit between the 3D structures of squid and bovine rhodopsin extends to the location of a short, transverse a-helix at the cytoplasmic surface of cephalopod rhodopsin, which appears to be part of the intermolecular docking domain [74]. Helix VIII is located close to a site at which the C-terminus of rhodopsin may interact with the phospholipid bilayer via S-palmitoylation of two adjacent cysteine residues. These cysteine residues are conserved in many, but not all, invertebrate rhodopsins, as well as in vertebrate rhodopsins [14,79,80]. Moreover, mutation of the cysteines present at the C-terminus of Drosophila Rhl (see Figure 4) is without functional consequences (Bentrop unpublished).

Two other sites for post-translational modifications are observed in all invertebrate rhodopsins. All rhodopsins harbour at their extracellularly located N-terminal peptide at least one consensus sequence for N-glycosylation. On the cytoplasmic side, serine and threonine residues are located close to the C-terminus, which may serve as sites for multiple phosporylation by a receptor kinase. As indicated in Figure 4, there is no clear cut indication for a positional conservation, either of the sites for N-glycosylation or of the putative phosphorylation sites.

Post-translational N-glycosylation of invertebrate rhodopsin has been demonstrated for cephalopod rhodopsin [81] as well as for insect rhodopsin [82,83], Site-directed mutagenesis of the putative glycosylation sites in Drosophila Rhl indicated that only Asn 20 (see Figure 4), but not a second putative site (Asn 169), becomes glycosylated. The mutation (N20L) leads to the accumulation of a nascent state of rhodopsin and causes photoreceptor degeneration [83,84], possibly by disturbing the interaction of nascent rhodopsin with the chaperone NinaA [85,86]. The functional importance of rhodopsin glycosylation for folding, sorting, and transport is highlighted by the finding that Rhl adopts the gylcosylated state only transiently. Thus, while cephalopod (Octopus) rhodopsin remains equipped with a rather unique oligosaccharide side chain after being incorporated into the rhabdomeral photoreceptor membrane, mature Rhl rhodopsin in the rhabdomeric membrane of fly photoreceptors is deglycosylated [68,83]. For Octopus rhodopsin it has been recently shown by mass spectrometry that in addition to the N-glycan, which is conserved in 7TM receptors, two iV-acetylgalactosamine residues are O-linked near the N-terminus [87], The functional relevance of these glycosylations remains to be elucidated.

Phosphorylation of vertebrate rhodopsin was recognized as the first light-triggered enzymatic modification of any rhodopsin [88-90], It has been shown subsequently that multiple phosphorylation of vertebrate rhodopsin at C-terminally located serine residues is an essential step in the deactivation of the light-activated rhodopsin state. Phosphorylation of the active state (Mil) leads to a rapid high-affinity interaction of the regulator protein, arrestin, with Mil, which hinders Mil from further activating the G-protein, transducin, (see Chapter 3). Thus, in the vertebrate visual cycle, rhodopsin phosphorylation is directly linked to the function of arrestin in regulating the active state of rhodopsin.

Cephalopod rhodopsin [91,92] and fly rhodopsin [93-96] also undergo a light-dependent phosphorylation-dephosphorylation cycle (Figure 5). In these rhodopsins, a single site (Octopus [97]) or multiple sites (flies [94]) become light-dependently phosphorylated and dephosphorylated. The light-dependent steps in this phosphorylation cycle are induced by the conversion of rhodopsin (P-state) into metarhodopsin (M-state). Conversion of P into M also induces the binding of Arrestin2 (Arr2), one of the arrestin isoforms expressed in photoreceptors (Figure 5). Dephosphorylation of M is induced by photoregeneration of M into P, as a result of the release of arrestin, which allows the phosphorylated M to interact with protein phosphatase [94], Thus, a link between Arr2 function and light-activated phosphorylation of rhodopsin in fly photoreceptors exists at the level of the regulation of rhodopsin dephosphorylation. The distinct and profound difference regarding the function of rhodopsin phosphorylation in fly photoreceptors is that here phosphorylation per se is not a prerequisite for binding of arrestin to the active M-state. This is supported by the finding that Arr2 still binds to the M-state of Drosophila Rhl and suppresses the activity of metarhodopsin even if the C-terminal peptide containing the phosphorylation sites is removed [98].

Activation of Gq

Conversion ofRhodopsin to Metarhodopsin

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