Erythroid spectrin is the predominant component of the twodimensional protein network called the membrane skeleton, underlying the lipid bilayer of red cells (for recent reviews, seeRefs. 1-3). Formation of the membrane skeleton involves multiple protein-protein interactions among integral membrane proteins. Interactions of spectrin with other membrane proteins such as ankyrin, protein 4.1, and adducin provide a linkage of spectrin either to the plasma membrane or among spectrin tetramers. Many hereditary anemia mutations affect interactions of these integral membrane proteins, resulting in increased fragility and shortened lifespan of erythrocytes. In hereditary elliptocytosis and pyropoikilocytosis, the mutations have been localized in the ␣-and ␤-subunits of spectrin (reviewed in Refs. 4 and 5). Many of these proteins, including spectrin, which were first identified in red cells, have isoforms expressed in nonerythroid cells, but the structure and regulatory processes of the nonerythroid membrane skeleton are less well understood (reviewed in Refs. 1-3, 6, and 7). Functional differences between the membranes of erythroid and nonerythroid cells argue against the simple erythrocyte model of the membrane skeleton. Major differences between the erythroid model and other cells include differences in the expression of spectrin (8 -11) and ankyrin isoforms (12-15) (reviewed in Ref.16), interactions of spectrin and ankyrin with additional proteins (17-21), localization of spectrin in the cytoplasm as well as in the plasma membrane (10,11,22), and the potential for dramatic rearrangements of spectrin's cellular location (23, 24) (reviewed in Refs. 2 and 7).Several studies have demonstrated that both erythroid and nonerythroid spectrins are expressed in brain tissue (8 -11, 25). Neuronal compartmentalization of brain spectrin isoforms into axons and presynaptic terminals (nonerythroid spectrin) and into cell bodies and dendrites (erythroid spectrin) (10, 25) suggests that brain spectrin isoforms may perform related but distinct functions in neuronal cells. It has been suggested that nonerythroid spectrin performs a more general, constitutive role, while erythroid spectrin takes part in more specialized activities of differentiated cells (26). The ␣-subunit of erythroid spectrin, ␣I (27), 1 and the ␣-subunit of nonerythroid spectrin, ␣II (28, 29), each contains a unique SH3 2 domain. Distinct protein interactions are likely to involve these domains, and they may be important for specific distribution and specialized roles of brain spectrin isoforms.

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The gene encoding human spectrin Src homology domain binding protein 1, or Hssh3bp1, which is a marker of macropinocytic vesicles and a potential regulator of macropinocytosis, co-localizes to a YAC containing chromosome 10p sequences at loci D10S89 and D10S111 that are frequently deleted in prostate tumors. Expression of Hssh3bp1 was evaluated at the protein level in 17 paired normal and malignant prostate tumor samples using the monoclonal antibody 2G8 to Hssh3bp1. These experiments demonstrated that 4/6 tumors (67%) with 10p deletion failed to express Hssh3bp1 protein compared to 5/11 (46%) tumors with intact 10p. Thus, loss of Hssh3bp1 expression is concordant with allelic loss of adjacent 10p sequences in human prostate tumors. In addition, two prostate tumor cell lines contain an exon skipping mutation in the Hssh3bp1 gene that leads to the abnormal splicing of the mRNA and loss of a portion of Abl tyrosine kinase SH3 domain binding site in the protein. These data are consistent with a role for Hssh3bp1 as a candidate tumor suppressor gene inactivated during prostate tumorigenesis.

The majority of familial Alzheimer's disease (AD) cases are linked to mutations on presenilin 1 and 2 genes (PS1 and PS2). The normal function of the proteins and the mechanisms underlying early-onset AD are currently unknown. To address this, we screened an expression library for proteins that bind differentially to the wild-type PS1 and mutant in the large cytoplasmic loop (PS1L

The cytoplasmic domain of the beta amyloid precursor protein (betaAPP) may play a role in cellular events that lead to the secretion of the Abeta peptide, the major constituent of amyloid plaques found in the brains of individuals affected by Alzheimer's disease, by interacting with cellular factors involved in betaAPP function or processing. In order to elucidate the structural basis of cytoplasmic domain activity, the conformations adopted in solution by a peptide containing the 47-residue C-terminal sequence of betaAPP have been investigated by NMR and CD spectroscopy. The peptide does not have a stable tertiary structure, but local regions of the polypeptide chain populate defined conformations. In particular, the amino acid sequences TPEE and NPTY form type I reverse turns. These structured regions correspond to sequences within the cytoplasmic domain implicated in the biological activity of betaAPP.

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Spectrins represent a family of membrane-associated proteins responsible for membrane flexibility and cell shape in erythrocytes, and probably in most nonerythroid cells. Spectrin functions as a tetramer consisting of two heterodimers each containing two subunits termed alpha and beta. In humans, alphaI and alphaII spectrins but not beta spectrins are characterized by the presence of an Src homology 3 (SH3) domain. As a tool to investigate the function of spectrin SH3 domains we derived several monoclonal antibodies (mAb) to the recombinant human alphaI or alphaII spectrin SH3 domain. Immunostaining using these monoclonal antibodies indicated expression of alphaI spectrin in cell bodies and alphaII spectrin in neurites of granule neurons in mouse primary cerebellar cultures. Monoclonal antibodies reactive to alphaI spectrin SH3 domain indicated expression of a protein(s) containing an alphaI-like SH3 domain in cytoplasmic vesicular-like structures in GFAP-positive cells in these cultures. In NIH 3T3 fibroblasts, these antibodies label macropinocytic vesicles. Together, these data and Western blotting results suggest expression of at least three spectrin-SH3 domain antibody-reactive proteins.