Adducins are a family of cytoskeleton proteins encoded by three genes (␣, , ␥). In a comprehensive assay of gene expression, we show the ubiquitous expression of ␣-and ␥-adducins in contrast to the restricted expression of -adducin. -adducin is expressed at high levels in brain and hematopoietic tissues (bone marrow in humans, spleen in mice). To elucidate adducin's role in vivo, we created -adducin null mice by gene targeting, deleting exons 9-13. A 55-kDa chimeric polypeptide is produced from the first eight exons of -adducin and part of the neo cassette in spleen but is not detected in peripheral RBCs or brain. -adducin null RBCs are osmotically fragile, spherocytic, and dehydrated compared with the wild type, resembling RBCs from patients with hereditary spherocytosis. The lack of -adducin in RBCs leads to decreased membrane incorporation of ␣-adducin (30% of normal) and unexpectedly promotes a 5-fold increase in ␥-adducin incorporation into the RBC membrane skeleton. This study demonstrates adducin's importance to RBC membrane stability in vivo.Adducin was originally described as a protein kinase C substrate in RBCs (1). Purified human RBC adducin consists of two similar polypeptides, ␣ (M r of 103,000) and  (M r of 97,000) (2). In vitro, RBC adducin crosslinks actin filaments with spectrin in a Ca 2ϩ -calmodulin-dependent manner (3), and bundles (4) and caps (5) actin filaments. Adducin is also a substrate for kinase (6, 7) and protein kinase A (8). The third member of the adducin family, ␥-adducin, was discovered as a protein kinase C binding protein in kidney (9). ␥-adducin, a doublet of 84,000 and 86,000 M r , interacts with ␣-adducin in kidney cell extracts (9). Alternatively spliced mRNAs have been described from all three adducin genes (10, 11). Most encode truncated isoforms compared with the originally described isoforms, and their functions are not yet known. To determine the function of -adducin in vivo, we created a null mutation in mice. -adducin null RBCs are osmotically fragile and demonstrate properties similar to RBCs from patients with hereditary spherocytosis. This study demonstrates adducin's importance in RBC structure in vivo.
MATERIALS AND METHODSTargeted Disruption of the -Adducin Gene (Add2). The targeting vector was constructed in the pPNT (12) plasmid by using a 3.9-kilobase (kb) EcoRI-BamHI fragment as the 5Ј homology segment and a 1.9-kb EcoRI-NotI fragment as the 3Ј homology segment (Fig. 3A). Transfected 129͞Sv-derived J1 embryonic stem cells (12) were cultured and selected in G418 and gancyclovir. Genomic DNA was digested with EcoRV and was analyzed by Southern blotting using a flanking EcoRVEcoRI fragment as the hybridization probe (Fig. 3B). Blastocyst injection and embryo transfer were performed by using standard techniques (13). Male chimeras were mated to C57BL͞6J females to generate heterozygotes. Progeny were genotyped by using PCR on tail biopsies.Red Blood Cell Analysis. Blood counts were determined by using a Technicon H3 analyzer (Bayer Diagnosti...
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