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Abstract
The rod outer segment guanylyl cyclase 1 (ROS-GC1) is an essential component of photo-transduction in the retina. In the light-induced signal cascade, membrane-bound ROS-GC1 restores cGMP levels in the dark in a calcium-dependent manner. With decreasing calcium concentration in the intracellular compartment, ROS-GC1 is activated via the intracellular site by guanylyl cyclase-activating proteins (GCAP-1/-2). Presently, the exact activation mechanism is elusive. To obtain structural insights into the ROS-GC1 regulation by GCAP-2, chemical cross-linking/mass spectrometry studies using GCAP-2 and three ROS-GC1 peptides were performed in the presence and absence of calcium. The majority of cross-links were identified with the C-terminal lobe of GCAP-2 and a peptide comprising parts of ROS-GC1's catalytic domain and C-terminal extension. Consistently with the cross-linking results, surface plasmon resonance and fluorescence measurements confirmed specific binding of this ROS-GC peptide to GCAP-2 with a dissociation constant in the low micromolar range. These results imply that a region of the catalytic domain of ROS-GC1 can participate in the interaction with GCAP-2. Additional binding surfaces upstream of the catalytic domain, in particular the juxtamembrane domain, can currently not be excluded.
Highlights
The retinal guanylyl cyclase 1 (ROS-GC1) is a transmembrane protein enabling the light adaption process in the eye’s rods and cones (Koch, 1991; Dizhoor et al, 1994; Goraczniak et al, 1994)
We studied an N-terminally extended version of peptide 1 comprising a part of the catalytic domain in order to gain more detailed insights into the Guanylyl cyclase-activating protein (GCAP)-2 binding site at rod outer segment guanylyl cyclase 1 (ROS-GC1)
The majority of cross-links were obtained with ROS-GC1 peptide 2, in which two segments reacted preferably
Summary
The retinal guanylyl cyclase 1 (ROS-GC1) is a transmembrane protein enabling the light adaption process in the eye’s rods and cones (Koch, 1991; Dizhoor et al, 1994; Goraczniak et al, 1994). Low calcium concentrations result in the activation of ROS-GC1 by the guanylyl cyclase-activating proteins 1/2 (GCAP-1/-2) that bind to the 68-kDa intracellular ROS-GC1 domain (Koch et al, 2002). This process is known as phototransduction (Schwartz, 1985; Pugh and Cobbs, 1986). The review by Schwartz (1985) addresses biophysical and electrophysiological properties of the, at that time so-called, “light-sensitive current.”
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