Abstract
In retinal rod photoreceptor cells, transducin (Gt) 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 GDP-bound transducin exclusively reduced the extraction of negatively charged phosphatidylserine. The effect disappeared upon activation of the G-protein with guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). PDE affected the extraction of the zwitterionic phosphatidylcholine and, to a smaller extent, of phosphatidylethanolamine. When active GtGTPgammaS 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.
Highlights
The transduction of the light signal in the retinal rod photoreceptor is a well studied model system for G-protein-coupled signal transduction
Activation of G-protein transducin (Gt) by guanosine 5-O-(3-thiotriphosphate) (GTP␥S) resulted in a substantial dissociation of Gt from the membranes (Fig. 1, lane 4), whereas Gt activation in the presence of PDE enhanced membrane binding of PDE (Fig. 1, lane 5)
bovine serum albumin (BSA) Extraction of Spin-labeled Phospholipid Analogues—To identify and characterize the interaction of Gt and PDE with spin-labeled phospholipid analogues (SL-PL), we measured the BSA-extractable amounts of those analogues from disk membranes in the absence or presence of proteins
Summary
Transducin; BSA, fatty acid-free bovine serum albumin; BTP, 1,3-bis[tris(hydroxymethyl)methylamino]propane buffer; Gt␣, ␣-subunit of transducin; Gt␥, ␥ heterodimer subunit of transducin; GTP␥S, guanosine 5Ј-O-(3-thiotriphosphate) bound; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; PDE, cGMP-phosphodiesterase; ROS, rod outer segment; SL-PC, 1-palmitoyl-2-(4-doxylpentanoyl) phosphatidylcholine; SL-PE, 1-palmitoyl-2-(4-doxylpentanoyl) phosphatidylethanolamine; SL-PS, 1-palmitoyl-2-(4-doxylpentanoyl) phosphatidylserine; SL-PL, spin-labeled phospholipids. Heterotrimeric Gt is peripherally attached to the disk membrane by weak hydrophobic and ionic interactions Both Nterminal acylation of the Gt␣-subunit and C-terminal farnesylation of the Gt␥-subunit are required for membrane association of Gt [4]. This study provides direct information on the phospholipid interaction of the signaling proteins in native disk membranes. We are interested in the dynamics of this proteinlipid interaction during activation of the signal cascade To characterize this interaction, we incorporate spin-labeled phospholipids into disk membranes, and we probe their accessibility to extraction by bovine serum albumin (BSA) [15, 16]. Anchoring of Signal Proteins to Specific Lipid Head Groups spin-labeled phospholipid analogues from the disk membrane in the absence or presence of Gt and PDE, both in the dark and under conditions of light activation. Gt anchors in a small cluster of PS, whereas PDE binds to a large “cushion” of more than 70 Ϯ 10 molecules of phosphatidylcholine (PC) and phosphatidylethanolamine (PE)
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