Signal Transduction in the Visual Cascade Involves Specific Lipid-Protein Interactions

Abstract

In retinal rod photoreceptor cells, transducin (G t ) and cyclic GMP phosphodiesterase (PDE) are peripherally anchored to the cytoplasmic surface of the disk saccules. We have examined the role of specific phospholipids in the interaction of these proteins with native osmotically intact disk vesicles, employing spin-labeled phospholipid analogues (2% of total phospholipids) and bovine serum albumin back-exchange assay. Inactive GDPbound transducin exclusively reduced the extraction of negatively charged phosphatidylserine. The effect disappeared upon activation of the G-protein with guanosine 5-O-(3-thiotriphosphate) (GTP␥S). PDE affected the extraction of the zwitterionic phosphatidylcholine and, to a smaller extent, of phosphatidylethanolamine. When active G t GTP␥S interacted with the PDE to form the active effector, the interaction with phosphatidylcholine was specifically enhanced. Each copy of the G-protein bound 3 ؎ 1 molecules of phosphatidylserine, whereas the PDE bound a much larger amount (70 ؎ 10) of a mixture of phosphatidylcholine and ethanolamine. The results are interpreted as a head group-specific and state-dependent interaction of the signaling proteins with the phospholipids of the photoreceptor membrane.The transduction of the light signal in the retinal rod photoreceptor is a well studied model system for G-protein-coupled signal transduction. It involves the sequential activation of rhodopsin, transducin (G t ), 1 and cGMP phosphodiesterase (PDE) on the cytoplasmic surface of the disk saccules that fill the rod outer segment (ROS). Absorption of light transforms rhodopsin into the activated metarhodopsin II state, which then interacts with G t to rapidly catalyze the exchange of GDP for GTP in the nucleotide-binding site of the G t ␣-subunit. G t ␣-GTP dissociates and couples to the effector PDE (1, 2). Activation of PDE results in a decrease in the cytosolic cGMP concentration which in turn leads to closure of cGMP-regulated channels in the plasma membrane, hyperpolarization, and neuronal signaling (see Ref. 3).Heterotrimeric G t is peripherally attached to the disk membrane by weak hydrophobic and ionic interactions. Both Nterminal acylation of the G t ␣-subunit and C-terminal farnesylation of the G t␥ -subunit are required for membrane association of G t (4). Electrostatic interactions, especially of G t ␤␥, further enhance the membrane binding to negatively charged surfaces or to vesicles containing the acidic lipid phosphatidylserine (5-7). Inactive transducin in its GDP-bound form is attached as a G t ␣␤␥ heterotrimer to the membrane. G t couples to activated rhodopsin, triggering GDP release as a first step of catalytic nucleotide exchange. The interaction with the receptor occurs directly from the membrane-bound state of the holoprotein. After activation of transducin, G t ␣ dissociates from G t ␤␥ and couples stoichiometrically to the effector PDE.The PDE is a heterotetrameric protein composed of the undissociable ␣␤ complex (8) and two identical ␥-subunits (9). Full activation of ...

Electrostatic and Lipid Anchor Contributions to the Interaction of Transducin with Membranes

“…The tilted orientations suggested by Hessel et al and Chabre and le Maire (24,25) are similar to the G t orientation identified by our analysis as the most favorable. On the other hand, parallel orientations (16 -23) place the negatively charged patches around the C terminus of G t ␣ close to the negatively charged bilayer, making these orientations less favorable by ϳ2 kcal/mol (ϳ30-fold difference in occupancy probability).…”

“…In some orientations the N-terminal helix of G t ␣ was placed parallel to the membrane ("parallel orientations") (16 -23), whereas in others this helix was placed at a large angle with respect to the membrane plane ("tilted orientations") (24,25). In addition to their lipid anchors, G t and its individual subunits can interact electrostatically with the negatively charged membrane surface (16,18), and these interactions can affect both the affinity of transducin for the membrane and its orientation on the membrane surface.…”

Dynamics of Arrestin-Rhodopsin Interactions

“…For example, PS, which undergoes a light-dependent translocation to the cytoplasmic side of the membrane (39), may interact specifically with arrestin in the native ROS as it appears to do with transducin (54). Intriguingly phosphoinositide has been implicated in the trafficking of Drosophila visual arrestin and recruitment of ␤-arrestin to clathrin-coated pits, although…”

Lipid second messengers and related enzymes in vertebrate rod outer segments

“…The cGMP-gated cation channel of ROS was found to be inhibited by PI(4,5)P 2 ( 58 ). Specifi c interactions between phospholipids and proteins involved in phototransduction such as PC, PE, and PDE6 and between PS and Gt ␣ were observed ( 59 ). Among phospholipid classes, DHA-PA was found to display the highest binding to rhodopsin, thus suggesting a possible modulatory role of free DHA and DHA-PA in visual transduction ( 60 ).…”