Chapter 6

Transducin: Structure, function and role in phototransduction

The pineal and pituitary–adrenocortical secretions play an important role in adaptive responses of the organism acting as coordinating signals for both several biological rhythms and multiple neuroendocrine and metabolic functions.

The more relevant neuroendocrine changes occurring with ageing affect the secretion of melatonin and of corticosteroids. These changes may be clearly appreciated by the study of their circadian rhythmicity.

The circadian profile of plasma melatonin was clearly flattened in elderly subjects and even more in old individuals with dementia. Indeed, the impairment of melatonin signal occurring in aging was related either to age itself or to the cognitive performances of subjects.

The biosynthetic dissociation between glucocorticoids and androgen secretion is responsible for the selective impairment of androgens, such as DHEA and DHEA-S, by comparison to cortisol.

Due to the opposite effects of the two kinds of corticosteroids either in the periphery and in the CNS, the imbalance between glucocorticoids and androgens, well demonstrated by the evaluation of the cortisol/DHEA-S molar ratio, may be responsible for the occurrence in the CNS of a more neurotoxic steroidal milieu, already present in clinically healthy elderly subjects and especially in patients with dementia. The effects of that steroidal milieu are more prominent at the level of the hippocampal–limbic structure, involved both in the modulation of endocrine structures, such as the HPA axis, and in the control of cognitive, behavioral and affective functions.

Phototransduction in the vertebrate rod and cone photoreceptors is regulated by structurally homologous and yet distinct groups of signaling proteins. We have previously identified in bovine retinas a cone-specific G-protein γ subunit (G_γc, previously named G_γ8), which may play a key role in coupling the cone visual pigment to phosphodiesterase (O. C. Onget al.,1995,J. Biol. Chem.270: 8495–8500). We report here the characterization of human G_γcand its gene structure. Human G_γcsubunit shares a high degree of sequence identity with the corresponding bovine G_γcisoform (85%) and human rod G_γ1(63%). The protein is specifically localized in cones, as indicated by immunohistochemical staining using anti-G_γcantibodies. Nucleotide sequence analysis of the G_γcgene (GNGT2) reveals a structure consisting of three exons and two introns, with the intron splice sites similar to that of the rod G_γ1gene (GNGT1). By using fluorescencein situhybridization, we have further localized the human GNGT2 gene to chromosome 17q21. The elucidation of the G_γcgene structure would facilitate the identification of genetic defects associated with cone degeneration.

The phototransduction process in cones has been proposed to involve a G protein that couples the signal from light-activated visual pigment to the effector cyclic GMP phosphodiesterase. Previously, we have identified and purified a Gβγ complex composed of a Gβ₃isoform and an immunochemically distinct Gγ subunit (Gγ₈) from bovine retinal cones (Fung, B. K.-K., Lieberman, B. S., and Lee, R. H. (1992) J. Biol. Chem. 267, 24782-24788; Lee, R. H., Lieberman, B. S., Yamane, H. K., Bok, D., and Fung, B. K.-K. (1992a) J. Biol. Chem. 267, 24776-24781). Based on the partial amino acid sequence of this cone Gγ₈, we screened a bovine retinal cDNA library and isolated a cDNA clone encoding Gγ₈. The cDNA insert of this clone includes an open reading frame of 207 bases encoding a 69-amino acid protein. The predicted protein sequence of Gγ₈shares a high degree of sequence identity (68%) with the Gγ (Gγ₁) subunit of rod transducin. Similar to rod Gγ₁, it terminates in a CIIS motif that is the site for post-translational modification by farnesylation. Messenger RNA for Gγ₈is present at a high level in the retina and at a very low level in the lung, but is undetectable in other tissues. Immunostaining of bovine retinal sections with an antipeptide antibody against the N-terminal region of Gγ₈further shows a differential localization of Gγ₈to cones with a pattern indistinguishable from that of Gβ₃. This finding suggests that Gβ₃γ₈is a component of cone transducin involved in cone phototransduction and color vision.

When light strikes a rod photoreceptor cell in the retina, rhodopsin molecules become photoexcited and a series of biochemical events rapidly follows. Photoexcited rhodopsin activates a G protein, transducin, which in turn activates a cyclic guanosine monophosphate (cGMP)-phosphodiesterase (PDE). The resulting hydrolysis of cGMP leads to the closure of cation channels in the plasma membrane, which triggers a neural signaling event. This chapter focuses on rhodopsin and discusses the way it functions as a photoreceptor protein. This is of intrinsic importance because it forms the molecular basis of the visual transduction process. It is of general significance because rhodopsin is a well-studied member of the class of G protein-linked receptors. Thus, the function of rhodopsin in signal transduction may be of importance in understanding the behavior of the entire class of receptors. Two types of photoreceptor cells, rods and cones, are present in the vertebrate retina. Rod cells operate optimally at low light intensities and contain the photosensitive pigment rhodopsin. Cones operate at higher light intensities and facilitate color vision in many animals.

Transducin is the retinal rod outer segment (ROS)-specific G protein coupling the photoexcited rhodopsin to cyclic GMP-phosphodiesterase. The α subunit of transducin is known to be ADP-ribosylated by bacterial toxins. We investigated the possibility that transducin is modified in vitro by an endogenous ADP-ribosyltransferase activity. By using either ROS, cytosolic extract of ROS or purified transducin in the presence of [α??P]nicotinamide adenine dinucleotide (NAD*), the α and β subunits of transducin were found to be radiolabeled, The labeling was decreased by snake venom phosphodiesterase I (PDE 1). The modification was shown to be mono ADP-ribosylation by analyses on thin layer chromatography of the PDE 1-hydrolyzed products which revealed only 5′AMP residues. In addition we report that sodium nitroprusside activates the ADP-ribosylation of transducin.

Previous studies have shown that vertebrate rod outer segments (ROS) have a light activated phospholipase C which hydrolyzes phosphatidylinositol-4,5-bisphosphate (PIP₂). Three different experimental approaches have been used to test the hypothesis that the phosphatidylinositol (PI) biosynthetic cycle is present in ROS and that PIP₂ can be regenerated from DG independent of rod inner segments. In the first study, enzyme activities of the PI cycle were assayed simultaneously in the presence of CTP, myo-inositol and [γ-³²P]ATP using endogenous lipids as substrates. Under these conditions, broken (leaky) ROS prepared by continuous sucrose gradient centrifugation showed PI, PIP and DG kinase activities similar to those found in intact ROS and non-ROS membranes, whereas PI synthetase activity was much lower in the leaky ROS than in the other two fractions. The relative distribution of PI synthetase specific activity in the three membrane preparations was similar to that of the microsomal enzyme marker cytochrome c reductase. ROS prepared by discontinuous sucrose gradient centrifugation showed only 2–3% of whole homogenate PI synthetase or phosphatidyl: cytidyl transferase activities, and the distribution of activities was the same as for microsomal and mitochondrial marker enzymes. In the second study, whole retinas were incubated with myo-[2-³H]inositol or [2-³H]glycerol in vitro, and the time course of incorporation of radioactivity into PI and other phospholipids was determined for ROS and three other retinal fractions. Over a 10-hr period, the rate of incorporation of myo-[2-³H]inositol or [2-³H]glycerol into PI in ROS was lowest among the various retinal fractions. In the third study, chemical analysis of the molecular species composition of PI, DG and phosphatidic acid (PA) from ROS shows that PA is substantially different from PI and DG, the latter two being quite similar. These results are consistent with a precursor-product relationship between PI and DG, but not with the conversion of DG to PA or of PA to PI. Taken together, these three studies indicate that ROS do not have PI synthetase or phosphatidyl: cytidyl transferase activities, but do have DG, PI and PIP kinase activities. Thus, the PI in ROS lost through rapid turnover must be replaced with molecules derived from de novo synthesis in the inner segment of the photoreceptor cell.