Structure and binding of the C-terminal segment of R9AP to lipid monolayers.

Abstract

Phototransduction cascade takes place in disc membranes of photoreceptor cells. Following its activation by light, rhodopsin activates the G-protein transducin causing the dissociation of its GTP-bound α-subunit, which in turn activates phosphodiesterase 6 (PDE6) leading to the hyperpolarization of photoreceptor cells. PDE6 must then be inactivated to return to the dark state. This is achieved by a protein complex which is presumably anchored to photoreceptor disc membranes by means of the transmembrane C-terminal segment of RGS9-1-Anchor Protein (R9AP). Information on the secondary structure and membrane binding properties of the C-terminal segment of R9AP is not yet available to further support its role in the membrane anchoring of this protein. In the present study, circular dichroism and infrared spectroscopy measurements have allowed us to determine that the C-terminal segment of human and bovine R9AP adopts an α-helical structure in solution. Moreover, this C-terminal segment has shown affinity for most of the phospholipids typical of photoreceptor membranes. In fact, the physical state and the type of phospholipid as well as electrostatic interactions influence the binding of the human and bovine peptides to phospholipid monolayers. In addition, these measurements revealed that the human peptide has a high affinity for saturated phosphocholine, which may suggest a possible localization of R9AP in photoreceptor microdomains. Accordingly, infrared spectroscopy measurements have allowed determining that the C-terminal segment of R9AP adopts an ordered α-helical structure in the presence of saturated phospholipid monolayers. Altogether, these data are consistent with the typical α-helical secondary structure and behavior observed for transmembrane segments and with the proposed role of membrane anchoring of the C-terminal segment of human and bovine R9AP.