Defects in protein folding and trafficking are a common cause of photoreceptor degeneration, causing blindness. Photoreceptor cells present an unusual challenge to the protein folding and transport machinery due to the high rate of protein synthesis, trafficking and the renewal of the outer segment, a primary cilium that has been modified into a specialized light-sensing compartment. Phototransduction components, such as rhodopsin and cGMP-phosphodiesterase, and multimeric ciliary transport complexes, such as the BBSome, are hotspots for mutations that disrupt proteostasis and lead to the death of photoreceptors. In this chapter, we review recent studies that advance our understanding of the chaperone and transport machinery of phototransduction proteins.

Phosducin (Pdc), a highly conserved phosphoprotein involved in the regulation of retinal phototransduction cascade, transcriptional control, and modulation of blood pressure, is controlled in a phosphorylation-dependent manner, including the binding to the 14-3-3 protein. However, the molecular mechanism of this regulation is largely unknown. Here, the solution structure of Pdc and its interaction with the 14-3-3 protein were investigated using small angle x-ray scattering, time-resolved fluorescence spectroscopy, and hydrogen-deuterium exchange coupled to mass spectrometry. The 14-3-3 protein dimer interacts with Pdc using surfaces both inside and outside its central channel. The N-terminal domain of Pdc, where both phosphorylation sites and the 14-3-3-binding motifs are located, is an intrinsically disordered protein that reduces its flexibility in several regions without undergoing dramatic disorder-to-order transition upon binding to 14-3-3. Our data also indicate that the C-terminal domain of Pdc interacts with the outside surface of the 14-3-3 dimer through the region involved in Gtβγ binding. In conclusion, we show that the 14-3-3 protein interacts with and sterically occludes both the N- and C-terminal Gtβγ binding interfaces of phosphorylated Pdc, thus providing a mechanistic explanation for the 14-3-3-dependent inhibition of Pdc function.

Opening of G-protein-activated inward-rectifying K(+) (GIRK, Kir3) channels is regulated by interaction with βγ-subunits of Pertussis-toxin-sensitive G proteins upon activation of appropriate GPCRs. In atrial and neuronal cells agonist-independent activity (I(basal)) contributes to the background K(+) conductance, important for stabilizing resting potential. Data obtained from the Kir3 signaling pathway reconstituted in Xenopus oocytes suggest that I(basal) requires free G(βγ). In cells with intrinsic expression of Kir3 channels this issue has been scarcely addressed experimentally. Two G(βγ)-binding proteins (myristoylated phosducin - mPhos - and G(αi1)) were expressed in atrial myocytes using adenoviral gene transfer, to interrupt G(βγ)-signaling. Agonist-induced and basal currents were recorded using whole cell voltage-clamp. Expression of mPhos and G(αi1) reduced activation of Kir3 current via muscarinic M(2) receptors (IK(ACh)). Inhibition of IK(ACh) by mPhos consisted of an irreversible component and an agonist-dependent reversible component. Reduction in density of IK(ACh) by overexpressed Gαi1, in contrast to mPhos, was paralleled by substantial slowing of activation, suggesting a reduction in density of functional M2 receptors, rather than G(βγ)-scavenging as underlying mechanism. In line with this notion, current density and activation kinetics were rescued by fusing the αi1-subunit to an Adenosine A(1) receptor. Neither mPhos nor G(αi1) had a significant effect on I(basal), defined by the inhibitory peptide tertiapin-Q. These data demonstrate that basal Kir3 current in a native environment is unrelated to G-protein signaling or agonist-independent free G(βγ). Moreover, our results illustrate the importance of physiological expression levels of the signaling components in shaping key parameters of the response to an agonist.

The G-protein-coupled receptor (GPCR) signaling system is one of the main signaling pathways in eukaryotes. Here, we analyze the evolutionary history of all its components, from receptors to regulators, to gain a broad picture of its system-level evolution. Using eukaryotic genomes covering most lineages sampled to date, we find that the various components of the GPCR signaling pathway evolved independently, highlighting the modular nature of this system. Our data show that some GPCR families, G proteins, and regulators of G proteins diversified through lineage-specific diversifications and recurrent domain shuffling. Moreover, most of the gene families involved in the GPCR signaling system were already present in the last common ancestor of eukaryotes. Furthermore, we show that the unicellular ancestor of Metazoa already had most of the cytoplasmic components of the GPCR signaling system, including, remarkably, all the G protein alpha subunits, which are typical of metazoans. Thus, we show how the transition to multicellularity involved conservation of the signaling transduction machinery, as well as a burst of receptor diversification to cope with the new multicellular necessities.