Introductory Review Cellscience Reviews Vol 2 No.2 ISSN 1742-8130

The mGluR6 receptors in the retina: Analysis of a unique G-protein signaling pathway

Duvoisin R.M., Morgans CW & Taylor WR

Parallel processing in the retina

Similar to other sensory modalities, the visual system resolves information into several parallel processing streams. These parallel pathways are first generated within the retina, and are evident as an increase in functional diversity of cell types at the first and second synapses. The photoreceptors in the retina produce a point by point representation of the visual field. At each point, changes in the light intensity are encoded as graded polarizations in the membrane potential. This simple neural image is conveyed to the second order neurons as fluctuations in continuous glutamate release from the photoreceptor terminals (Tachibana et al., 1993), with increases in light intensity hyperpolarizing photoreceptors and suppressing glutamate release. The second order neurons, called bipolar cells, receive the photoreceptor signals through glutamate receptors in their dendrites. There are 10 distinct morphological and functional classes of bipolar cells (Boycott and Wässle, 1991; Euler and Wässle, 1995; Ghosh et al., 2004; MacNeil et al., 2004; Pignatelli and Strettoi, 2004), representing 10 parallel neural images of the visual field. These can be divided into two broad groups according to the sign of their responses. Off-bipolar cells produce excitatory signals when the local light intensity is lower than the mean or background intensity. On-bipolar cells produce excitatory signals when the local light intensity is higher than background. This functional difference arises by virtue of the glutamate receptors expressed within the dendrites of the On- and Off-bipolar cells, and the integrity of these pathways is maintained throughout the visual system (Schiller et al., 1986). The evolution of the On-bipolar cell mGluR6 signal transduction pathway represents a unique specialisation which confers a high degree of visual acuity over a wide range of light intensities.

Off-bipolar cells express ionotropic glutamate receptors, while On-bipolar cells express metabotropic receptors. The ionotropic glutamate receptors (iGluR) in Off bipolar cells gate excitatory non-selective cation channels. There is some evidence that the type of iGluR is specific for the subclass of Off cone bipolar cell, a factor that might be important in establishing different temporal tuning properties (DeVries, 2000; Freed, 2000). There have been reports in some species of glutamate receptors in On-bipolar cells that activate inhibitory potassium and chloride channels (Nawy and Copenhagen, 1987; Hirano and MacLeish, 1991; Grant and Dowling, 1996). It is more usual for the glutamate receptor in On-bipolar cells to be the metabotropic mGluR6 receptor, which is coupled to an excitatory cation channel; however, in contrast to iGluRs, mGluR6 activation closes the channel (Wilson et al., 1987; Yamashita and Wässle, 1991).

Thus mGluR6 receptors mediate an inhibitory response to glutamate, which provides the synaptic sign-inversion required to change the light-evoked hyperpolarization of photoreceptors into an excitatory, depolarising response in On-bipolar cells. All On-bipolar cells express the same mGluR6 receptor, and therefore differences in the temporal tuning of these cells is not regulated by glutamate receptor expression (but see Awatramani and Slaughter, 2000). There is evidence, however, for differences in the calcium sensitivity of the mGluR6 signal transduction cascade between rod and cone On-bipolar cells (Berntson and Taylor, 2000; Berntson et al., 2004a), which could introduce functional diversity (see below). The biochemical cascade that links mGluR6 receptor activation to channel gating is poorly understood, and the molecular identity of the cation channel is unknown. In the following we will review our current understanding of mGluR6 signalling.

Metabotropic glutamate receptors

The diversity of glutamate receptors (GluRs) is extensive, larger than for any other neurotransmitter receptor family. There are two broad classes of GluRs: ionotropic and metabotropic (for reviews see Nakanishi, 1992; Hollmann and Heinemann, 1994). Ionotropic GluRs are heteromeric proteins assembled from probably four subunits, encoded by related gene families. The subunit composition defines several ligand-gated ion channels, originally identified by the agonists NMDA, AMPA and kainate. In contrast, metabotropic glutamate receptors (mGluRs) are not channels themselves, but are coupled through trimeric GTP-binding proteins (G proteins) to second messenger pathways that can ultimately activate ion channels downstream.

It is now thought that mGluRs are dimers, with each subunit predicted to span the plasma membrane seven times (Romano et al., 1996). This structure is common to the G protein-coupled receptor (GPCR) superfamily, which includes opsins, and adrenergic and muscarinic receptors (reviewed in Pin and Duvoisin, 1995; Conn and Pin, 1997; Bockaert and Pin, 1999). Eight distinct metabotropic receptors termed mGluR1 through mGluR8 have been cloned (also named GRM1-GRM8 by the Human Gene Nomenclature Committee), and can be classified into three groups according to amino acid sequence comparisons, signal transduction mechanism, and pharmacological profiles. Group-I receptors, comprising mGluR1 and mGluR5, are coupled to the stimulation of phospholipase C (PLC), which leads to an increase in phosphoinositide turnover, and the release of Ca²⁺ from internal stores. The signalling pathways for group-II (mGluR2 and mGluR3) and group-III (mGluR4, -6, -7, -8) receptors are less well understood. In transfected fibroblasts, group-II and group-III receptors are coupled to the inhibition of adenylyl cyclase. Group-III receptors can be distinguished pharmacologically from other mGluRs by their sensitivity to the agonist L-(+)-2-Amino-4-phosphonobutyric acid (APB, also called L-AP-4), and the antagonists (S)-2-Amino-2-methyl-4-phosphonobutanoic acid (MAP4) and (RS)-?-Methylserine-O-phosphate (MSOP). With the exception of the mGluR6 isoform, group-II and group-III receptors are widely distributed in the CNS. The mGluR6 isoform is unique to the visual system, where it is precisely targeted to the dendritic tips of On-bipolar cells in the retina (Nomura et al., 1994; Vardi et al., 2000).

While cGMP may not directly gate the channel, it does strongly potentiate the light-evoked current. Snellman and Nawy (2004) simulated light responses in mouse On-bipolar cells by bathing the retinal slice in the mGluR6 agonist, L-AP-4, and then applying the group-II/group-III antagonist, CPPG, to activate an inward current. They found that the current was increased by inclusion of cGMP in the electrode and even further increased by inclusion of cGMP plus IBMX. This effect was minimal for long puffs of CPPG (mimicking a bright flash that saturates the postsynaptic response), but dramatic for short puffs (mimicking a dim flash, which produces partial deactivation of the mGluR6 receptors). Because inhibitors of cGMP-dependent protein kinases (cGKs) suppress cGMP-induced potentiation, the target of cGMP is most likely a cGK. Interestingly, a consensus cGK phosphorylation site is present in the C-terminal domain of mGluR6. Snellman and Nawy propose that phosphorylation by cGK reduces the efficacy by which glutamate-bound mGluR6 keeps the channel closed. This would result in an amplification of the current in response to dim light flashes, which cause only a small decrease in glutamate.

mGluR6 Signal Transduction

Much of what is known about GPCR-mediated signal transduction has been learned from studying the mechanisms of vertebrate phototransduction (reviewed by Arshavsky et al., 2002). Briefly, light-activated rhodopsin (or cone opsin) is coupled to transducin, a heteromeric GTP-binding protein (G-protein), which dissociates into a GTP-bound Gαt and a Gβγ dimer. The GTP-Gαt subunit stimulates a phosphodiesterase (PDE), which hydrolyses cGMP. Depletion of cGMP closes cGMP-gated cation channels (CNG channels) in the plasma membrane and hyperpolarizes the photoreceptor. This cascade of linked reactions can produce large signal amplification, which is exemplified in rod photoreceptors where a single photon produces hydrolysis of 10⁶ cGMP molecules and closes enough cGMP-gated channels to produce ~100µV hyperpolarization. Phosphorylation of rhodopsin by rhodopsin-kinase and the subsequent binding of arrestin terminate phototransduction. Following the release of all-trans-retinal, protein phosphatase 2A (PP2A) dephosphorylates rhodopsin, 11-cis-retinal binds and rhodopsin is ready for a new cycle. Gαt is inactivated by its intrinsic GTPase activity, which is enhanced by interacting with the PDEγ subunit and the regulator of G-protein signalling (RGS) protein, RGS9.

Early physiological studies suggested that the mGluR6 signal transduction cascade is analogous to phototransduction in photoreceptors. In patch-clamp recordings from dogfish, salamander, and cat On-bipolar cells, addition of cGMP to the recording electrode solution led to the development of an inward current, consistent with the activation of cGMP-gated channels (Nawy and Jahr, 1990; Shiells and Falk, 1990, 1992a; de la Villa et al., 1995; Walters et al., 1998). Shiells & Falk have provided a body of evidence indicating that mGluR6 transduction in dog-fish is similar to phototransduction (Shiells and Falk, 1990, 1992b, 1994). Moreover, Henry et al. (2003) have localized the mRNA for the cone CNG channel to On-bipolar cells in the goldfish retina, but these findings have not yet been confirmed by immunohistochemical analysis. Contrary to the results in fish, it seems certain that the cone CNG channel is not involved in mGluR6 signal transduction in other species. All rod bipolar cells in mammals are mGluR6-expressing On-bipolar cells, and knock-out of the cone CNG α subunit in mouse blocks cone-mediated responses, without affecting the rod pathway (Biel et al., 1999). Moreover, mutations in the human cone CNG α or β subunits selectively abolishes cone-mediated vision, leaving rod vision intact (Kohl et al., 1998; Kohl et al., 2000). Finally, physiological evidence from salamander and mouse suggest that hydrolysis of cGMP may not be an obligatory step in the mGluR6 pathway. On-bipolar cells responses persist in the presence of non-hydrolyzable cGMP analogues (Snellman and Nawy, 2004) and recent data suggests that responses to agonist may not be abolished by PDE inhibitors such as IBMX (Nawy, 1999) and dipyridamole as first believed (Nawy and Jahr, 1991; Nawy and Jahr, 1990; Shiells and Falk, 1990, 1992a; de la Villa et al., 1995; Walters et al., 1998). A transgenic mouse which does not express a functional cGMP PDE activity will be needed to finally establish whether the mGluR6 cascade is mediated via the activation of a PDE or through direct interaction with the cation channel.

While cGMP does not directly gate the channel, it does strongly potentiate the light-evoked current. Snellman and Nawy (2004) simulated light responses in mouse On-bipolar cells by bathing the retinal slice in the mGluR6 agonist, L-AP-4, and then applying the group-II/group-III antagonist, CPPG, to activate an inward current. They found that the current was increased by inclusion of cGMP in the electrode and even further increased by inclusion of cGMP plus IBMX. This effect was minimal for long puffs of CPPG (mimicking a bright flash that saturates the postsynaptic response), but dramatic for short puffs (mimicking a dim flash, which produces partial deactivation of the mGluR6 receptors). Because inhibitors of cGMP-dependent protein kinases (cGKs) suppress cGMP-induced potentiation, the target of cGMP is most likely a cGK. Interestingly, a consensus cGK phosphorylation site is present in the C-terminal domain of mGluR6. Snellman and Nawy propose that phosphorylation by cGK reduces the efficacy by which glutamate-bound mGluR6 keeps the channel closed. This would result in an amplification of the current in response to dim light flashes, which cause only a small decrease in glutamate.

Putative downstream components of the mGluR6 signaling pathway

Gαo: G proteins activated by 7 transmembrane-GPCRs, such as mGluR6, are trimeric proteins composed of α, β and γ subunits. Evidence is strong that the Gα subunit of the G protein coupled to mGluR6 is Gαo. Gαo dialyzed into On-bipolar cells occludes their glutamate response, and antibodies against Gαo reduce their glutamate response (Nawy, 1999). These results are supported by immunohistochemical labelling for Gαo, indeed a specific splice variant, Gαo1, in On-bipolar cells, including their dendrites in the OPL (Vardi et al., 1993; Dhingra et al., 2002; Huang et al., 2003). Moreover, recombinant mGluR6 activates Go 18 times more efficiently than transducin, the phototransduction G-protein, in an in vitro reconstitution system (Weng et al., 1997). Finally, electroretinograms (ERGs) of mice deficient in Gαo lack the b-wave, reflecting a block in synaptic transmission between photoreceptors and On-bipolar cells, (Dhingra et al., 2000), and in this respect are identical to mGluR6 knock-out mice (Masu et al., 1995).

Gαo is the most abundant heteromeric G-protein in the brain (Sternweis and Robishaw, 1984), yet there are no known endogenous downstream targets of Gαo. Mice in which the Gαo gene has been disrupted do not die at birth, indicating that Gαo is not necessary for life, but the mice rarely reach adulthood and have severe neurological problems including the abnormal ERG described above (Jiang et al., 1998).

The major components of the mGluR6 pathway, and their possible interactions are illustrated within the inset Figure.

Gβγ: One possible function of Gαo is that it may serve as a regulatory subunit for effects more directly mediated by the Gβγ dimers. Knock-out of Gαo is accompanied by a reduction of β subunits to 32% of wild type (Mende et al., 1998). Gβγ dimers are known activators of phospholipase C, K⁺ channels (GIRKS), and calcium channels (Herlitze et al., 1996); however, given that mGluR6 activation closes rather than opens ion channels, as usually reported for Gβγ dimers, the action of Gβγ in On-bipolar cells may be inhibitory (Blake et al. 2001). It is not known which β and γ subunits associate with Gαo in the mGluR6 pathway. A likely candidate is Gγ13, previously identified as part of the gustducin G-protein complex in taste cells (Huang et al., 1999). Antibodies against Gγ13 strikingly label On-bipolar cells in the mouse retina including their dendrites in the OPL (Huang et al., 2003). Several G-protein β subunits have also been localized in the OPL including Gβ3 and Gβ4 (Huang et al., 2003), which can form functional Gβγ dimers with Gγ13 (Blake et al., 2001).

Ret-RGS1: RGS proteins, or regulators of G-protein signalling, promote the GTP hydrolysis activity of the Gα subunit. A unique feature of the signal transduction pathway activated by mGluR6 is that membrane depolarization at light On results from a deactivation of the pathway. A limiting factor is likely GTP hydrolysis and it is most interesting that Ret-RGS1, a retina-specific variant of RGS20, was found to associate with Gαo. Moreover, Ret-RGS1 was immunolocalized to retinal synaptic layers, including the dendrites of On-bipolar cells (Faurobert et al., 1999; Dhingra et al., 2004). One possibility is that RGS1 accelerates hydrolysis of GTP, and thereby inactivates Gαo more rapidly and accelerates On-bipolar cell light responses.

L7: L7, also called Purkinje cell protein-2 (pcp2), is another protein that binds to Gαo (Luo and Denker, 1999; Natochin et al., 2001; Dhingra et al., 2004). This binding is mediated by a 19 amino acid so-called GoLoco (or G protein regulatory, GPR) motif. Proteins containing GoLoco motifs possess guanine nucleotide dissociation inhibitor (GDI) activity. Indeed, L7 has been shown to inhibit GDP release and GTP binding to Gαo and Gαi (Natochin et al., 2001), although Luo and Denker (1999) found the opposite effect. L7 is expressed only in rod bipolar cells and cerebellar Purkinje cells (Berrebi et al., 1991; Grünert and Martin, 1991). Rod bipolar cells are uniquely capable of relaying the drop in glutamate release from rod photoreceptors following single rhodopsin isomerizations (Field and Rieke, 2002; Berntson et al., 2004b). Thus L7 may regulate the signalling pathway activated by mGluR6 in rod bipolar cells by inhibiting the re-association of Gαo and the Gβγ dimer, thereby prolonging the effects of glutamate and producing saturation of the signal transduction pathway at lower glutamate concentrations.

Nyctalopin: Nyctalopin, a small leucine-rich repeat protein expressed in the retina, was originally identified as the site of mutations causing complete congenital stationary night blindness (CSNB1, Bech-Hansen et al., 2000; Pusch et al., 2000). ERGs from patients with CSNB1 lack a b-wave but have a normal a-wave consistent with a block in synaptic transmission between photoreceptors and On-bipolar cells. A naturally occurring animal model of CSNB1, the nob (or no b-wave) mouse (Pardue et al., 1998), contains a deletion in the mouse nyctalopin gene (Gregg et al., 2003b). Measurements of light responses in the inner retina of the nob mouse indicate a complete lack of On-responses whereas signalling through the cone Off pathway appears to be normal (Gregg et al., 2003a). Electrophysiological studies on retinal slices from the nob mouse found that the On-bipolar cells fail to respond to exogenously applied glutamate, whereas the Off-bipolar cells respond normally (Gregg et al., 2002). Interestingly, the immunolocalization of mGluR6 appears normal in the nob mouse retina (Ball et al., 2003). These findings suggest that nyctalopin is required for the On-bipolar cell response to glutamate, most likely acting downstream of the mGluR6 receptor.

mGluR6 receptors and single photon signaling

The potential for high signal transduction gain may be important for the physiological function of mGluR6 receptors in the rod pathway. In mammals, rod photoreceptors contact a specialized On-bipolar cell, the rod-bipolar cell. At the lowest light intensities, rod-bipolar cells signal the absorption of at most one or two photons, and the conditions required for this to occur have been reviewed in detail elsewhere (Field et al., 2004; Taylor and Smith, 2004). Briefly, a single photon in a rod must suppress glutamate release for long enough that the mGluR6 pathway becomes deactivated, and allows the cation channels in the rod-bipolar cell to open. On the other hand, the mGluR6 pathway should not react too readily otherwise random gaps in vesicle release from the rods might be falsely signalled as single photon signals. Thus it has been proposed that the mGluR6 pathway in rod-bipolar cells acts as a switch, turning off very rapidly when the pause in transmitter release exceeds some threshold duration, but remaining active for subthreshold durations that are not likely to be related to photon capture (van Rossum and Smith, 1998; Field and Rieke, 2002; Berntson et al., 2004b). Such non-linear behaviour is not required in the On-cone bipolar cells, which operate under conditions when photons are plentiful. Therefore it seems likely that the mGluR6 signalling pathways in rod and cone bipolar cells might display subtle functional differences related to their distinct physiological roles. For example, the L7 protein is present in rod-bipolar cells but not cone bipolar cells. As noted above, L7 may work to prolong the action of glutamate at the mGluR6 receptors, thereby slowing the postsynaptic mGluR6 response. This could extend temporal integration and prevent random gaps in vesicle release from erroneously signalling the detection of a photon by the rod.

Another difference between rod and cone bipolar cells relates to the calcium sensitivity of the mGluR6 pathway. Physiological recordings from mouse rod-bipolar cells show that synaptic transmission is strongly suppressed by influx of calcium through the mGluR6 channels in the dendritic tips (Berntson et al., 2004a). Similar postsynaptic calcium feedback has been documented in salamander and fish bipolar cells, although on a time-scale two orders of magnitude slower (Walters et al., 1998; Shiells, 1999; Shiells and Falk, 1999; Nawy, 2000). It has been suggested that the calcium feedback might mediate adaptation to increasing background light intensities. Further work will be required to establish the role of the feedback, and importantly, to determine the molecular mechanisms that allow incoming calcium ions to alter transduction through the mGluR6 pathway.

Conclusion

Despite its central importance in vision, the pathway by which light causes the depolarisation of On-bipolar cells remains to be fully elucidated. A number of the major constituents in the signalling pathway have been identified, although the final effector, the cation channel remains elusive. Identification of the cation channel would represent a major advance in solving this puzzle, as it would allow the pathway to be traced from both ends. There remains considerable speculation as to whether the light-regulated cation channel of the On-bipolar cell might belong to the CNG or TRP families, or even some novel class of ion channel, and its molecular identification is keenly anticipated.

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