Inositol-1,4,5-trisphosphate-binding proteins controlling the phototransduction cascade of invertebrate visual cells.

The main phototransduction cascade in invertebrate visual cells involves the turnover of phosphatidylinositol, an important biochemical mechanism common to many signal-transduction systems. Light-activated rhodopsin stimulates guanine nucleotide exchange on the Gq class of G-protein, which activates phospholipase C to hydrolyze phosphatidylinositol 4,5-bisphosphate to inositol-1,4,5-trisphosphate and diacylglycerol. Subsequently, inositol-1,4,5-trisphosphate-binding proteins continue the signal cascade. Here, we report on the first inositol-1,4,5-trisphosphate-binding proteins demonstrated in an invertebrate visual system with our investigation of the photosensitive rhabdoms of squid. We screened the ability of proteins to interact with inositol-1,4,5-trisphosphate by affinity column chromatography with an inositol-1,4,5-trisphosphate analogue. We detected an inositol-1,4,5-trisphosphate-binding affinity in phospholipase C, receptor kinase and five other proteins in the cytosolic fraction and, surprisingly, rhodopsin in the membrane fraction. A binding assay with (3)H-labelled inositol-1,4,5-trisphosphate demonstrated the inositol-1,4,5-trisphosphate affinity of each of the purified proteins. Since rhodopsin, receptor kinase and phospholipase C are involved upstream of phosphatidylinositol turnover in the signal cascade, our result suggests that phosphatidylinositol turnover is important in feedback pathways in the signalling system.

A possible role of RGS9 in phototransduction. A bridge between the cGMP-phosphodiesterase system and the guanylyl cyclase system.

In the current concept of phototransduction, the concentration of cGMP in retinal rod outer segments is controlled by the balance of two enzyme activities: cGMP phosphodiesterase (PDE) and guanylyl cyclase (GC). However, no protein directly mediates these two enzyme systems. Here we show that RGS9, which is suggested to control PDE activity through regulation of transducin GTPase activity (He, W., Cowan, C. W., and Wensel, T. G. (1998) Neuron 20, 95-102), directly interacts with GC. When proteins in the Triton X-100-insoluble fraction of bovine rod outer segments were isolated by two-dimensional gel electrophoresis and binding of GC to these proteins was examined using a GC-specific antibody, proteins (55 and 32 kDa) were found to interact with GC. However, the activity of GC bound to the 55-kDa protein was not detected. This observation was elucidated by the finding that the 55-kDa protein inhibited GC activity in a dose-dependent manner. Amino acid sequence showed that five peptides derived from the 55-kDa protein were identical to corresponding peptides of RGS9. Together with other biochemical characterization of the 55-kDa protein, these observations indicate that the 55-kDa protein is RGS9 and that RGS9 inhibits GC. RGS9 may serve as a mediator between the PDE and GC systems.

The traffic of particles in the axonic process of vertebrate cone-type photoreceptor cells.

Differential-interference-contrast microscopy with video enhancement displayed the movement of particles for the first time in the isolated axonic process of cone-type photoreceptor cells of Rana catesbiana. This movement was observed under visible light which visual pigments could absorb. The number of retrograde moving particles in an arbitrary area on the axonic process was twice that of those moving in the anterograde direction. The mean velocities were 1.03 +/- 0.55 microns/sec for anterograde particles and 0.41 +/- 0.30 microns/sec for retrograde particles, which are of the same order as those found in isolated neurons.

Regulation of G protein function by an effector in GTP-dependent signal transduction. An inhibitory subunit of cGMP phosphodiesterase inhibits GTP hydrolysis by transducin in vertebrate rod photoreceptors.

The regulation of cGMP phosphodiesterase in vertebrate rod photoreceptors is a typical G protein-dependent signal transduction mechanism. The interaction of P gamma, an inhibitory subunit of cGMP phosphodiesterase, with transducin alpha subunit (T alpha) is essential for the activation of cGMP phosphodiesterase. It has been shown that, in a homogenized preparation of frog (Rana catesbeiana) rods, P gamma interacts with GTP.T alpha and remains tightly bound to GDP.T alpha after GTP hydrolysis on T alpha. Association of this complex with beta gamma subunits of transducin (T beta gamma) triggers the release of P gamma from the complex and the subsequent inactivation of cGMP phosphodiesterase. In a system reconstituted with purified components, both GTP- and GDP-bound forms of T alpha were found to interact with P gamma. Under these conditions, P gamma inhibited GTP hydrolysis by transducin in a noncompetitive manner with a Ki of 92 nM. Binding of an hydrolysis-resistant GTP analog to T alpha was also inhibited by P gamma. These inhibitions of transducin function were resulted from the inhibition of both hydrolysis of GTP bound to T alpha and interaction of GDP.T alpha with membrane-bound T beta gamma. However, after GDP.T alpha reassociated with membrane-bound T beta gamma, the inhibitory effect of P gamma on the binding of an hydrolysis-resistant GTP analog to T alpha was greatly diminished, suggesting that the GTP/GDP exchange on T alpha was not inhibited by P gamma. These data indicate that the T alpha function is altered during complexing with P gamma. G protein functions may be modified by interacting with an effector in the G protein-dependent signal transduction.

Fourier transform infrared spectroscopic study on retinochrome and its primary photoproduct, lumiretinochrome.

Structural studies of retinochrome, and its photoproduct, lumiretinochrome, were done by Fourier transform infrared difference spectroscopy. The absorption bands in the carbonyl stretching region which shift in D2O show the changes in the protein part during the photoreaction. Strong absorption bands in the finger-print region show that the all-trans-retinal chromophore in retinochrome isomerizes to the 11-cis-retinal chromophore in lumiretinochrome upon illumination with yellow-green light at 83K.