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The interaction of human erythrocyte protein 4.1 (4.1R) with phosphatidylinositol-4,5-bisphosphate (PIP2) and the effect of PIP2 on the interaction of 4.1R with membrane proteins are characterized and assessed. The results have shown that PIP2 is a major, selective regulatory cofactor in red cell membrane-cytoskeletal interactions.

Mammalian red blood cells, unlike those of other vertebrates, must withstand the rigors of circulation in the absence of new protein synthesis. Key to this is plasma membrane elasticity deriving from the protein spectrin, which forms a network on the cytoplasmic face. Spectrin is a tetramer [([alpha][beta]).sub.2], made up of [alpha][beta] dimers linked head to head. We show here that one component of erythrocyte spectrin, [alpha]I, is encoded by a gene unique to mammals. Phylogenetic analysis suggests that the other [alpha]-spectrin gene ([alpha]II) common to all vertebrates was duplicated after the emergence of amphibia, and that the resulting [alpha]I gene was preserved only in mammals. The activities of al and [alpha]II spectrins differ in the context of the human red cell membrane. An [alpha]I-spectrin fragment containing the site of head-to-head interaction with the [beta]-chain binds more weakly than the corresponding [alpha]II fragment to this site. The latter competes so strongly with endogenous [alpha]I as to cause destabilization of membranes at 100-fold lower concentration than the [alpha]I fragment. The efficacies of [alpha]I/[alpha]II chimeras indicate that the partial structural repeat, which binds to the complementary [beta]-spectrin element, and the adjacent complete repeat together determine the strength of the dimer--dimer interaction on the membrane. Alignment of all available [alpha]-spectrin N-terminal sequences reveals three blocks of sequence unique to [alpha]I. Furthermore, human [alpha]II-spectrin is closer to fruitfly [alpha]-spectrin than to human [alpha]I-spectrin, consistent with adaptation of [alpha]I to new functions. We conclude that [alpha]I-spectrin represents a neofunctionalized spectrin adapted to the rapid make and break of tetramers. cytoskeleton | membrane | red blood cell | triple-helix

Synapsin proteins are implicated in synaptic maintenance and in vesicle supply for exocytosis. Synapsin I has been observed to bind and be regulated by calmodulin. It is now shown that a representative of synapsin II binds calmodulin. Calmodulin binding by synapsins is not confined to mammals and it is concluded that calmodulin binding to synapsins could be a general aspect of synaptic function regulation.