Interactions between integral membrane proteins and the cytoskeleton in red blood cells studied by measured molecular lateral diffusion
Creators: name: Knowles, David W.
Publisher: name: University of British Columbia
Year: 2008

description: Integral membrane receptor proteins are involved in many aspects of biological function. These proteins are restricted to the lipid bilayer but can move laterally within it and interact with specific agents which control their functional activity. The agents, collectively termed ligands, are commonly other molecules, which vary in size from solubilized ions to large macromolecules. Receptor/ligand interactions can influence lateral organization and conformation of receptor proteins and are the mechanism by which biological signals are passed across the membrane and from cell to cell. A single receptor protein embedded within a pure lipid bilayer has considerable lateral mobility, which is restricted only by the viscosity of the bilayer, and to a lesser extent, by the viscosity of the surrounding aqueous environment [Saffman &amp; Delbruck 1975]. Cell membranes however, are comprised of many receptor proteins as well as a network of support proteins which closely associate with the bilayer on its cytoplasmic side. Consequently, receptor-protein lateral mobility is considerably lower in membranes than in lipid bilayers, presumably because of weak non-covalent interactions with peripheral membrane components. Thus the cell membrane, like many biological systems, is a set of weakly and dynamically coupled components. To understand the nature of such dynamic coupling, the effect of receptor/ligand interactions on receptor lateral motion and membrane rigidity was studied in a model-cell system. The model system chosen was the red blood cell membrane because it contains several well characterized integral membrane proteins plus a peripheral cytoskeletal network. The major integral proteins are band 3 and several different types of glycophorin. The major cytoskeletal protein is a long (76-200nm) filamentous tetramer known as spectrin. The ligands used were monoclonal antibodies (divalent) and their Fab fragments (monovalent). The antibodies recognize specific epitopes on the extracellular domain of glycophorin A, the majorred cell sialoglycoprotein. Ligand 9A3 binds the most distal epitope of the extracellular do-main, ligands R10 and 10F7 bind to the mid-region of the extracellular domain and ligandB14 binds close to the lipid bilayer on the extracellular side of glycophorin A. Results from earlier studies [Chasis et al 1988] demonstrated that when these ligands (9A3,R10, 10F7 &amp; B14) are bound to the extracellular domain of glycophorin A, membrane deformability (measured by flow ectacytometry) was greatly reduced in normal red cells, but not in red cell variants that lack the cytoplasmic domain of glycophorin A (Miltenberger V red cells). Because the cytoskeletal network (rather than the lipid bilayer) is responsible for membrane rigidity, these results suggested that ligand binding can initiate a transmembrane signal that causes an increased interaction of the cytoplasmic domain of the receptor with the underlying cytoskeletal network. To test this hypothesis and to investigate the molecular features of such a mechanism, the lateral motion of red cell integral receptor proteins (glycophorin A, glycophorin C &amp;band 3) and the mechanical rigidity of the membrane were measured in relation to the amount of ligand (eg. 9A3, R10, 10F7 or B14) bound to the extracellular domain of glycophorin A in both normal and variant red blood cells. Lateral motion (lateral mobility and mobile fraction) of the integral proteins was measured in situ by the technique of fluorescence recovery after photo bleaching (FRAP). This technique involves: (1) quantitating the fluorescence intensity from labels conjugated to the ligand or receptor of interest on the membrane; (2) irreversibly bleaching a small region of the membrane and (3) measuring the subsequent fluorescence recovery due to lateral motion of fluorophore on the surface. Red cell membrane rigidity was measured by the method of pipette aspiration where a portion of the red cell membrane was deformed into a pipette by a minute suction pressure. The dependence of aspiration length on applied pressure is linear for elastic deformations and results in an effective rigidity modulus which is a combination of the elastic shear and the area expansion moduli of the membrane cytoskeleton. The findings are that anti-glycophorin A ligands (R10 &amp; B14), bound to normal red cell membranes, cause the immobilization of both glycophorin A and band 3, and produce a marked increase in membrane rigidity. These effects are due to an increased interaction between the cytoplasmic domain of the integral proteins and the cytoskeletal network. This was evident for several reasons: (1) the effect was independent of ligand valency, indicating that extracellular cross-linking was not responsible for the immobilization; (2) lateral immobilization and increased rigidity were not seen if ligand was bound to Miltenberger V red cells (In these cells the cytoplasmic domain of glycophorin A is effectively absent due to natural mutation); (3) only partial immobilization and a small increase in membrane rigidity was seen when ligand was bound to spherocytic red cells (These cells are characterized by a severe spectrin deficiency which produces gross defects in the normal hexagonal structure of the cytoskeleton). Immobilization of band 3 resulted only for anti-glycophorin A ligands R10 (10F7) and B14. These ligands bind close to the bilayer surface and produce the great-est increase in membrane rigidity. Ligand 9A3 however, had less of an immobilizing effecton glycophorin A, had little effect on membrane rigidity and did not immobilize band 3.In light of the strong correlation between increased membrane rigidity and immobilization of band 3, and because the cytoplasmic domain of glycophorin A is a small peptide, it is proposed that the cytoplasmic domain of ligand bound glycophorin A interacts (directly) with band 3 to cause immobilization of both integral proteins, and increased membrane rigidity. Finally, this proposed link between band 3 immobilization and the increased membrane rigidity was tested in the case of ovalocytic red cells which are characterized by a tolerance of malarial invasion, and an increased membrane rigidity. It has been long assumed that the high rigidity of these cells, which is responsible for their malaria parasite tolerance, is due to a mutation in one of the skeletal proteins. No such mutation has ever been found. However, this study shows that the entire band 3 population is immobile in these cells. A parallel collaborative study shows that ovalocytic band 3 contains a mutation, a deletion of 9 amino acids in its cytoplasmic domain [Mohandas et al 1992]. Thus, it is apparent that the interaction of band 3 with the underlying cytoskeleton plays a crucial role in the de-termination of membrane mechanical properties, and the lateral motion of integral receptor proteins, and serves as a useful model for the understanding of the molecular mechanisms involved in receptor protein function.