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Cone photoreceptor cells of night-migratory songbirds seem to process the primary steps of two different senses, vision and magnetoreception. The molecular basis of phototransduction is a prototypical G protein-coupled receptor pathway starting with the photoexcitation of rhodopsin or cone opsin thereby activating a heterotrimeric G protein named transducin. This interaction is well understood in vertebrate rod cells, but parameter describing protein-protein interactions of cone specific proteins are rare and not available for migratory birds. European robin is a model organism for studying the orientation of birds in the earth magnetic field. Recent findings showed a link between the putative magnetoreceptor cryptochrome 4a and the cone specific G-protein of European robin. In the present work, we investigated the interaction of European robin cone specific G protein and cytoplasmic regions of long wavelength opsin. We identified the second loop in opsin connecting transmembrane regions three and four as a critical binding interface. Surface plasmon resonance studies using a synthetic peptide representing the second cytoplasmic loop and purified G protein α-subunit showed a high affinity interaction with a KD value of 21 nM. Truncation of the G protein α-subunit at the C-terminus by six amino acids slightly decreased the affinity. Our results suggest that binding of the G protein to cryptochrome can compete with the interaction of G protein and non-photoexcited long wavelength opsin. Thus, the parallel presence of two different sensory pathways in bird cone photoreceptors is reasonable under dark-adapted conditions or during illumination with short wavelengths. [ABSTRACT FROM AUTHOR]

The retinal degeneration protein RD3 is involved in regulatory processes of photoreceptor cells. Among its main functions is the inhibition of photoreceptor specific membrane guanylate cyclases during trafficking from the inner segment to their final destination in the outer segment. However, any physiological role of RD3 in non-retinal tissue is unsolved at present and specific protein targets outside of retinal tissue have not been identified so far. The family of membrane bound guanylate cyclases share a high homology of their amino acid sequences in their cytoplasmic domains. Therefore, we reasoned that membrane guanylate cyclases that are activated by natriuretic peptides are also regulated by RD3. We analyzed transcript levels of the rd3 gene and natriuretic peptide receptor genes Npr1 and Npr2 in the mouse retina, cerebellum, hippocampus, neocortex, and the olfactory bulb during development from the embryonic to the postnatal stage at P60. The rd3 gene showed a lower expression level than Npr1 and Npr2 (encoding for GC-A and GC-B, respectively) in all tested brain tissues, but was at least one order of magnitude higher in the retina. RD3 and natriuretic peptide receptor GCs co-express in the retina and brain tissue leading to functional tests. We expressed GC-A and GC-B in HEK293T cells and measured the inhibition of GCs by RD3 after activation by natriuretic peptides yielding inhibitory constants around 25 nM. Furthermore, endogenous GCs in astrocytes were inhibited by RD3 to a similar extent. We here show for the first time that RD3 can inhibit two hormone-stimulated GCs, namely GC-A and GC-B indicating a new regulatory feature of these hormone receptors. [ABSTRACT FROM AUTHOR]

Surface plasmon resonance (SPR) spectroscopy is a technique to study protein-protein interactions in real time; however, application of SPR spectroscopy for investigations of membrane receptors is difficult with respect to functional and uniform immobilization of receptors on a biosensor surface. In the current study, we developed a simple, direct, biosensor-based approach to monitor the molecular interactions between G protein transducin ([G.sub.t]) and rhodopsin (Rho), a prototypical G protein-coupled receptor (GPCR). Detergent-solubilized dark-adapted Rho was captured onto a biosensor surface via lectin interaction, enabling site-directed immobilization of the receptor that made its cytoplasmic surface accessible to a coupling G protein. The system resembled the natural system with respect to receptor density, binding of [G.sub.t] following flash or constant light application, fast GTP-dependent dissociation of [G.sub.t] from Rho, regeneration of Rho, and dependence of [G.sub.t] binding on light intensity and on concentration of [G.sub.t]. The apparent KD of the [G.sub.t]/Rho interaction was 13.6 nM. Our results validate the use of SPR spectroscopy as a tool to study G protein activation in GPCR systems and could be extended for application to other interaction partners of GPCRs.