The outer segment of vertebrate photoreceptors is a specialized compartment that hosts all the signaling components required for visual transduction. Specific to rod photoreceptors is an unusual set of three glutamic acid-rich proteins (GARPs): two soluble forms, GARP1 and GARP2, and the N-terminal cytoplasmic domain (GARP' part) of the B1 subunit of the cyclic GMP-gated channel.
In rod outer segments, GARP proteins have been proposed to control subcellular propagation of Ca2+ signals from the Ca2+-entry site, the cyclic nucleotide-gated channel, to the cytosol. GARP1 and GARP2 can be regarded as loose coils with low-affinity, high-capacity Ca2+ binding. Thus, GARPs might be involved in photoreceptor adaptation, which is controlled by Ca2+. Importantly, the GARP' part of the CNG channel may function as a molecular wire that is "channeling" Ca2+ from the exit site of the channel onto the surface of the disk and thereby compartmentalises Ca2+ microdomains.
GARPs have been shown to interact with proteins at the rim of the disc membrane. Amino acid sequence analysis of GARPs revealed structural features typical of "natively unfolded" proteins. By using biophysical techniques, including size-exclusion chromatography, dynamic light scattering, NMR spectroscopy, and circular dichroism, we showed that GARPs indeed exhibit a large degree of intrinsic disorder.
Analytical ultracentrifugation and chemical cross-linking showed that GARPs exist in a monomer/multimer equilibrium. The results suggested that the function of GARP proteins is linked to their structural disorder. They may provide flexible spacers or linkers tethering the cyclic GMP-gated channel in the plasma membrane to peripherin at the disc rim to produce a stack of rings of these protein complexes along the long axis of the outer segment. GARP proteins could then provide the environment needed for protein interactions in the rim region of discs.
We aim to analyse the protein interactions at the rim region on the single-molecule level using biochemical and biophysical techniques including cryo-electron microscopy, scanning electron microscopy (SEM), and high-resolution transmission electron microscopy (TEM).
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