Calcium-sensitive ROS-GC1 signaling outside of photoreceptors: A common theme

Until now, ROS-GC1 signal transduction system was thought to be exclusive to photoreceptors in the retina. Two recent reports, however, now show that this is not the case. In one, the ROS-GC1 signal transduction system has been identified and characterized in pinealocyte neurons. This signaling is modulated by norepinephrine. However, the response of the individual pinealocyte neuron to the norepinephrine signal depends on whether the GCAP1-linked (results in hyperpolarization) or S100-linked (results in depolarization) pathway is operational in the pinealocyte. The GCAP1-linked pathway results in hyperpolarization, while the S100-linked pathway, in depolarization. The two pathways are mutually exclusive. In the other report, the calcium-modulated ROS-GC1:GCAP1 signaling system has been discovered in mitral cells of the olfactory bulb. These findings raise the possibility that a common theme of calcium-modulated ROS-GC signaling may be utilized in a wide variety of neurosensory cells. This idea is also supported from evolutionary and functional perspectives.     

Distinct ONE-GC transduction modes and motifs of the odorants: Uroguanylin and CO2

In a subset of the olfactory sensory neurons ONE-GC^$ membrane guanylate cyclase is a central component of two odorant-dependent cyclic GMP signaling pathways. These odorants are uroguanylin and CO₂. The present study was designed to decipher the biochemical and molecular differences between these two odorant signaling mechanisms. The study shows (1) in contrast to uroguanylin, CO₂ transduction mechanism is Ca²⁺-independent. (2) CO₂ transduction site, like that of uroguanylin-neurocalcin δ, resides in the core catalytic domain, aa 880-1028, of ONE-GC. (3) The site, however, does not overlap the signature neurocalcin δ signal transduction domain, ⁹⁰⁸LSEPIE⁹¹³. Finally, (4) this study negates the prevailing concept that CO₂ uniquely signals ONE-GC activity (Sun et al. [19]; Guo et al. [21]). It demonstrates that it also signals the activation of photoreceptor membrane guanylate cyclase ROS-GC1. These results show an additional new transduction mechanism of the membrane guanylate cyclases and broaden our understanding of the molecular mechanisms by which different odorants using a single guanylate cyclase can regulate diverse cyclic GMP signaling pathways.     

Rod outer segment membrane guanylate cyclase type 1 (ROS-GC1) gene: Structure,organization and regulation by phorbol ester,a protein kinase C activator

At present there are two recognized members of the ROS-GC subfamily of membrane guanylate cyclases. They are ROS-GC1 and ROS-GC2. A distinctive feature of this family is that its members are not switched on by the extracellular peptide hormones; instead, they are modulated by intracellular Ca2+ signals, consistent to their linkage with phototransduction. An intriguing feature of ROS-GC1, which distinguishes it from ROS-GC2, is that it has two Ca2+ switches. One switch inhibits the enzyme at micromolar concentrations of Ca2+, as in phototransduction; the other, stimulates. The stimulatory switch, most likely, is linked to retinal synaptic activity. Thus, ROS-GC1 is linked to both phototransduction and the synaptic activity. The present study describes (1) the almost complete structural identity of 18.5 kb ROS-GC1 gene; (2) its structural organization: the gene is composed of 20 exons and 19 introns with classical GT/AG boundaries; (3) the activity of the ROS-GC1 promoter assayed through luciferase reporter in COS cells; and (4) induction of the gene by phorbol ester, a protein kinase C (PKC) activator. The co-presence of PKC and ROS-GC1 in photoreceptors suggests that regulation of the ROS-GC1 gene by PKC might be a physiologically relevant phenomenon.     

S100B-modulated Ca2+-dependent ROS-GC1 transduction machinery in the gustatory epithelium: a new mechanism in gustatory transduction

Gustatory transduction is a biochemical process by which the gustatory signal generates the electric signal. The microvilli of the taste cells in the gustatory epithelium are the sites of gustatory transduction. This study documents the biochemical, molecular, and functional identity of the Ca2+-modulated membrane guanylate cyclase transduction machinery in the bovine gustatory epithelium. The machinery is a two-component system: the Ca2+-sensor protein, S100B; and the transducer, ROS-GC1. S100B senses increments in free Ca2+, undergoes conformational change, binds to the domain amino acids (aa) Gly962-Asn981 and via the transduction domain aa Ile1030-Gln1041 activates ROS-GC1, generating the second messenger, cyclic GMP. In a recent study, operational presence of this machinery has been demonstrated in the photoreceptor bipolar synapse [Duda et al., EMBO J. 21 (2002) 2547]. Thus, the machinery has a broader role in sensory perceptions, vision in the retinal neurons and gustation in the tongue. The entry of the ROS-GC transduction machinery defines the beginning of a new paradigm of Ca2+ signaling in the tongue.     

WTAPELL motif of the photoreceptor ROS-GC1: A general phototransduction switch

This study documents the identity of an intriguing transduction mechanism of the [Ca²⁺]_i signals by the photoreceptor ROS-GC1. Despite their distal residences and operational modes in phototransduction, the two GCAPs transmit and activate ROS-GC1 through a common Ca²⁺ transmitter switch (Ca²⁺TS). A combination of immunoprecipitation, fluorescent spectroscopy, mutational analyses and reconstitution studies has been used to demonstrate that the structure of this switch is ⁶⁵⁷WTAPELL⁶⁶³. The two Ca²⁺ signaling GCAP pathways converge in Ca²⁺TS, get transduced, activate ROS-GC1, generate the LIGHT signal second messenger cyclic GMP and yet functionally perform divergent operations of the phototransduction machinery. The findings define a new Ca²⁺-modulated photoreceptor ROS-GC transduction model; it is depicted and discussed for its application to processing the different shades of LIGHT.     

First 3D-Structural Data of Full-Length Guanylyl Cyclase 1 in Rod-Outer-Segment Preparations of Bovine Retina by Cross-Linking/Mass Spectrometry

The rod-outer-segment guanylyl cyclase 1 (ROS-GC1) is a key transmembrane protein for retinal phototransduction. Mutations of ROS-GC1 correlate with different retinal diseases that often lead to blindness. No structural data are available for ROS-GC1 so far. We performed a 3D-structural analysis of native ROS-GC1 from bovine retina by cross-linking/mass spectrometry (XL-MS) and computational modeling. Absolute quantification and activity measurements of native ROS-GC1 were performed by MS-based assays directly in bovine retina samples. Our data present the first 3D-structural analysis of active, full-length ROS-GC1 derived from bovine retina. We propose a novel domain organization for the intracellular domain ROS-GC1. Our XL-MS data of native ROS-GC1 from rod-outer-segment preparations of bovine retina agree with a dimeric architecture. Our integrated approach can serve as a blueprint for conducting 3D-structural studies of membrane proteins in their native environment.     

Bicarbonate Modulates Photoreceptor Guanylate Cyclase (ROS-GC) Catalytic Activity

By generating the second messenger cGMP in retinal rods and cones, ROS-GC plays a central role in visual transduction. Guanylate cyclase-activating proteins (GCAPs) link cGMP synthesis to the light-induced fall in [Ca²⁺]_i to help set absolute sensitivity and assure prompt recovery of the response to light. The present report discloses a surprising feature of this system: ROS-GC is a sensor of bicarbonate. Recombinant ROS-GCs synthesized cGMP from GTP at faster rates in the presence of bicarbonate with an ED₅₀ of 27 mm for ROS-GC1 and 39 mm for ROS-GC2. The effect required neither Ca²⁺ nor use of the GCAPs domains; however, stimulation of ROS-GC1 was more powerful in the presence of GCAP1 or GCAP2 at low [Ca²⁺]. When applied to retinal photoreceptors, bicarbonate enhanced the circulating current, decreased sensitivity to flashes, and accelerated flash response kinetics. Bicarbonate was effective when applied either to the outer or inner segment of red-sensitive cones. In contrast, bicarbonate exerted an effect when applied to the inner segment of rods but had little efficacy when applied to the outer segment. The findings define a new regulatory mechanism of the ROS-GC system that affects visual transduction and is likely to affect the course of retinal diseases caused by cGMP toxicity.     

Characterization of the myristoyl lipid modification of membrane-bound GCAP-2 by H solid-state NMR spectroscopy

Guanylate cyclase-activating protein-2 (GCAP-2) is a retinal Ca²⁺ sensor protein. It is responsible for the regulation of both isoforms of the transmembrane photoreceptor guanylate cyclase, a key enzyme of vertebrate phototransduction. GCAP-2 is N-terminally myristoylated and full activation of its target proteins requires the presence of this lipid modification. The structural role of the myristoyl moiety in the interaction of GCAP-2 with the guanylate cyclases and the lipid membrane is currently not well understood. In the present work, we studied the binding of Ca²⁺-free myristoylated and non-myristoylated GCAP-2 to phospholipid vesicles consisting of dimyristoylphosphatidylcholine or of a lipid mixture resembling the physiological membrane composition by a biochemical binding assay and ²H solid-state NMR. The NMR results clearly demonstrate the full-length insertion of the aliphatic chain of the myristoyl group into the membrane. Very similar geometrical parameters were determined from the ²H NMR spectra of the myristoyl group of GCAP-2 and the acyl chains of the host membranes, respectively. The myristoyl chain shows a moderate mobility within the lipid environment, comparable to the acyl chains of the host membrane lipids. This is in marked contrast to the behavior of other lipid-modified model proteins. Strikingly, the contribution of the myristoyl group to the free energy of membrane binding of GCAP-2 is only on the order of − 0.5 kJ/mol, and the electrostatic contribution is slightly unfavorable, which implies that the main driving forces for membrane localization arises through other, mainly hydrophobic, protein side chain-lipid interactions. These results suggest a role of the myristoyl group in the direct interaction of GCAP-2 with its target proteins, the retinal guanylate cyclases.