The interaction between membrane proteins and cytoplasmic structural proteins is thought to be one mechanism for maintaining the spatial order of proteins within functional domains on the plasma membrane. Such interactions have been characterized extensively in the human erythrocyte, where a dense, cytoplasmic matrix of proteins comprised mainly of spectrin and actin, is attached through a linker protein, ankyrin, to the anion transporter (Band 3). In several nonerythroid cell types, including neurons, exocrine cells and polarized epithelial cells homologues of ankyrin and spectrin (fodrin) are localized in specific membrane domains. Although these results suggest a functional linkage between ankyrin and fodrin and integral membrane proteins in the maintenance of membrane domains in nonerythroid cells, there has been little direct evidence of specific molecular interactions. Using a direct biological and chemical approach, we show here that ankyrin binds to the ubiquitous (Na+ + K+)ATPase, which has an asymmetrical distribution in polarized cells.
Cytoskeletal proteins associate with specific cell adhesion complexes and membrane proteins and influence the structural and functional organization of polarized epithelial cells in the kidney. Among such proteins that have been studied in cultured cell lines and in animals are the tight junction complex (ZO-1 and occludin), the adherens cell-cell adhesion complex (alpha-, beta-catenin and plakoglobin), and Na+,K+-ATPase, with its associated membrane skeleton proteins ankyrin and fodrin. Although abnormal distribution of these proteins has been implicated in the pathogenesis of various renal diseases, the relevance of these findings to corresponding disease of the human kidney remains to be established. As a first step towards elucidating a role for such proteins in human kidney disease, we undertook a histochemical analysis of the distribution of these proteins in biopsy specimens of human kidney taken from healthy kidney transplant donors. We found each protein to have a characteristic subcellular localization and an intensity of staining that varied among different segments of the nephron in a manner that is consistent with discrete, segmental nephron function.
The membrane-skeleton of adult chicken neurons in the cerebellum and optic system is composed of polypeptides structurally and functionally related to the erythroid proteins spectrin and ankyrin, respectively. Neuronal spectrin comprises two distinct complexes that share a common alpha subunit (Mr 240,000) but which have structurally distinct polymorphic subunits (beta' beta spectrin; Mr 220/225,000; gamma spectrin, Mr 235,000); the brain-specific form (alpha gamma spectrin or fodrin) and an erythrocyte-specific form (alpha beta' beta spectrin). Two structurally related isoforms of ankyrin have also been identified and are termed alpha (Mr 260,000) and beta (Mr 237,000) ankyrin. Immunofluorescence demonstrates that the variants of spectrin and ankyrin, respectively, have different distributions within neurons. On the one hand, alpha gamma spectrin and beta ankyrin are present throughout the neuron, in the perikaryon, dendrites, and axon, whereas alpha beta' spectrin and alpha ankyrin are localized exclusively in the perikaryon and dendrites where they are actively segregated from alpha gamma spectrin and other components of axonal transport. This asymmetric distribution of spectrin and ankyrin isoforms is established in distinct stages during neuronal morphogenesis. Early in cerebellar and retinal development, alpha gamma spectrin is expressed in mitotic cells. Subsequently beta ankyrin and alpha gamma spectrin are coexpressed in postmitotic cells and gradually accumulate on the plasma membrane in a uniform pattern throughout the neuron during the phase of cell growth. At the onset of synaptogenesis and the cessation of cell growth, their levels of synthesis decline sharply while the assembled proteins remained as stable membrane components. Concomitantly, there is a dramatic induction in the accumulation of alpha ankyrin and alpha beta' spectrin, whose assembly is limited to the plasma membrane of the perikarya and dendrites. These results demonstrate that two successive, developmentally regulated programs of ankyrin and spectrin expression and patterning on the plasma membrane are involved in the assembly of the spectrin-based asymmetry in the neuronal membrane-skeleton, and that their asymmetric distribution is actively maintained throughout the life of the neuron.
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