Protein 4.1: Static and dynamic aspects of its association with the human erythrocyte membrane
Abstract
Human erythrocyte protein 4.1 is a major membrane skeletal protein that connects the spectrin-actin network to the lipid bilayer via associations with glycophorins and the cytoplasmic domain of band 3. Protein 4.1 is essential for normal erythrocyte stability yet the modes of its interaction with the membrane are only partially understood. No information is available regarding this protein's binding sites on the cytoplasmic domain of band 3 or about cellular factors that regulate its binding to membranes. We have mapped the protein 4.1 binding site on the cytoplasmic domain of band 3 utilizing radiolabel transfer cross linking, protease protection assays, and deletional mutagenesis techniques as well as antibody, peptide and ankyrin competition experiments. The predominant site of interaction resides in the N-terminus of band 3 between amino acid residues 1 and 46. In addition, evidence for a minor site near the junction of the cytoplasmic and membrane spanning domains was obtained. Dynamic regulation of membrane skeletal linkages is an important contribution to long term erythrocyte stability given the deformations and shear stress encountered during circulation. Calmodulin was demonstrated to block the association of protein 4.1 with membranes, an effect which is potentiated by free calcium. The calcium dependency of inhibition suggests that this mode of regulation occurs in pathological states of the erythrocyte where higher calcium concentrations prevail. A metabolite, 2,3 diphosphoglycerate, was shown to potently down regulate the binding of protein 4.1 to band 3 while partial blockage of ankyrin binding was also observed. Both of the described modes of inhibition measured in vitro occurred within physiological concentrations of calmodulin and 2,3 diphosphoglycerate. This implicates that such regulation will be prominent in vivo.
Degree
Ph.D.
Advisors
Low, Purdue University.
Subject Area
Biochemistry
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