We report the identification of βIV spectrin, a novel spectrin isolated as an interactor of the receptor tyrosine phosphatase-like protein ICA512. The βIV spectrin gene is located on human and mouse chromosomes 19q13.13 and 7b2, respectively. Alternative splicing of βIV spectrin generates at least four distinct isoforms, numbered βIVΣ1–βIVΣ4 spectrin. The longest isoform (βIVΣ1 spectrin) includes an actin-binding domain, followed by 17 spectrin repeats, a specific domain in which the amino acid sequence ERQES is repeated four times, several putative SH3-binding sites and a pleckstrin homology domain. βIVΣ2 and βIVΣ3 spectrin encompass the NH2- and COOH-terminal halves of βIVΣ1 spectrin, respectively, while βIVΣ4 spectrin lacks the ERQES and the pleckstrin homology domain. Northern blots revealed an abundant expression of βIV spectrin transcripts in brain and pancreatic islets. By immunoblotting, βIVΣ1 spectrin is recognized as a protein of 250 kD. Anti–βIV spectrin antibodies also react with two additional isoforms of 160 and 140 kD. These isoforms differ from βIVΣ1 spectrin in terms of their distribution on subcellular fractionation, detergent extractability, and phosphorylation. In islets, the immunoreactivity for βIV spectrin is more prominent in α than in β cells. In brain, βIV spectrin is enriched in myelinated neurons, where it colocalizes with ankyrinG 480/270-kD at axon initial segments and nodes of Ranvier. Likewise, βIV spectrin is concentrated at the nodes of Ranvier in the rat sciatic nerve. In the rat hippocampus, βIVΣ1 spectrin is detectable from embryonic day 19, concomitantly with the appearance of immunoreactivity at the initial segments. Thus, we suggest that βIVΣ1 spectrin interacts with ankyrinG 480/270-kD and participates in the clustering of voltage-gated Na+ channels and cell-adhesion molecules at initial segments and nodes of Ranvier.

Pleckstrin homology (PH) domains play a central role in a wide array of signaling pathways by binding second messenger lipids of the phosphatidylinositol phosphate (PIP) lipid family. A given type of PIP lipid is formed in a specific cellular membrane where it is generally a minor component of the bulk lipid mixture. For example, the signaling lipid PI(3,4,5)P 3 (or PIP 3 ) is generated primarily in the inner leaflet of the plasma membrane where it is believed to never exceed 0.02% of the bulk lipid. The present study focuses on the PH domain of the general receptor for phosphoinositides, isoform 1 (GRP1), which regulates the actin cytoskeleton in response to PIP 3 signals at the plasma membrane surface. The study systematically analyzes both the equilibrium and kinetic features of GRP1-PH domain binding to its PIP lipid target on a bilayer surface. Equilibrium binding measurements utilizing protein-to-membrane fluorescence resonance energy transfer (FRET) to detect GRP1-PH domain docking to membrane-bound PIP lipids confirm specific binding to PIP 3 . A novel FRET competitive binding measurement developed to quantitate docking affinity yields a K D of 50 ± 10 nM for GRP1-PH domain binding to membrane-bound PIP 3 in a physiological lipid mixture approximating the composition of the plasma membrane inner leaflet. This observed K D lies in a suitable range for regulation by physiological PIP 3 signals. Interestingly, the affinity of the interaction decreases at least 12-fold when the background anionic lipids phosphatidylserine (PS) and phosphatidylinositol (PI) are removed from the lipid mixture. Stopped-flow kinetic studies using protein-to-membrane FRET to monitor association and dissociation time courses reveal that this affinity decrease arises from a corresponding decrease in the on-rate for GRP1-PH domain docking with little or no change in the off-rate for domain dissociation from membrane-bound PIP 3 . Overall, these findings indicate that the PH domain interacts not only with its target lipid, but also with other features of the membrane surface. The results are consistent with a previously undescribed type of two-step search mechanism for lipid binding domains in which weak, nonspecific electrostatic interactions between the PH domain and background anionic lipids facilitate searching of the membrane surface for PIP 3 headgroups, thereby speeding the high-affinity, specific docking of the domain to its rare target lipid.Many significant events in cell signaling take place at membrane surfaces, particularly at the surface of the plasma membrane where receptors, channels, and signaling complexes make critical decisions to turn specific pathways on or off. An important class of membranebound second messengers that often play key roles in such decisions are the phosphatidylinositol phosphate lipids (PIP 1 lipids). Three of the most well characterized PIP signaling lipids in the plasma membrane are PI(3,4,5)P 3 , PI(3,4)P 2 , and PI(4,5)P 2 . These † Support provided by NIH Grant GM R01-63235 (to J.J...

Islet cell autoantigen (ICA) 512 is a novel autoantigen of insulin‐dependent diabetes mellitus (IDDM) which is homologous to receptor‐type protein tyrosine phosphatases (++PTPases). We show that ICA 512 is an intrinsic membrane protein of secretory granules expressed in insulin‐producing pancreatic beta‐cells as well as in virtually all other peptide‐secreting endocrine cells and neurons containing neurosecretory granules. ICA 512 is cleaved at its luminal domain and, following exposure at the cell surface, recycles to the Golgi complex region and is sorted into newly formed secretory granules. By immunoprecipitation, anti‐ICA 512 autoantibodies were detected in 15/17 (88%) newly diagnosed IDDM patients, but not in 10/10 healthy subjects. These results suggest that tyrosine phosphorylation participates in some aspect of secretory granule function common to all neuroendocrine cells and that a subset of autoantibodies in IDDM is directed against an integral membrane protein of insulin‐containing granules.

SummaryGlutamic acid decarboxylase (GAD) is the enzyme that synthesizes the neurotransmitter y-aminobutyric acid (GABA) in neurons and in pancreatic (3 cells. It is a major target of autoimmunity in Stiff Man syndrome (SMS), a rare neurological disease, and in insulin-dependent diabetes mellitus. The two GAD isoforms, GAD-65 and GAD-67, are the products of two different genes. GAD-67 and GAD-65 are very similar to each other in amino acid sequence and differ substantially only at their NH2-terminal region . We have investigated the reactivity of autoantibodies of 30 Stiff Man syndrome patients to GAD. All patient sera contained antibodies that recognize strongly GAD-65, but also GAD-67, when tested by immunoprecipitation on brain extracts and by immunoprecipitation or immunocytochemistry on cells transfected with either the GAD-65 or the GAD-67 gene. When tested by Western blotting, all patient sera selectively recognized GAD-65. Western blot analysis of deletion mutants of GAD-65 demonstrated that autoantibodies are directed predominantly against two regions of the GAD-65 molecule . All SMS sera strongly recognized a fragment contained between amino acid 475 and the COOH terminus (amino acid 585). Within this region, amino acids 475-484 and 571-585 were required for reactivity. The requirement of these two discontinuous segments implies that the epitope is influenced by conformation. This reactivity is similar to that displayed by the monoclonal antibody GAD 6, suggesting the presence of a single immunodominant epitope (SMS-E1) in this region of GAD-65 . In addition, most SMS sera recognized at least one epitope (SMS-E2) in the NH2-terminal domain of GAD-65 (amino acids 1-95) . The demonstration in SMS patients of a strikingly homogeneous humoral autoimmune response against GAD and the identification of dominant autoreactive target regions may help to elucidate the molecular mechanisms of GAD processing and presentation involved in GAD autoimmunity. Moreover, the reactivity reported here of GAD autoantibodies in SMS partially differs from the reactivity of GAD autoantibodies in insulin-dependent diabetes mellitus, suggesting a link between the pattern ofhumoral autoimmunity and the clinical condition .

Lateral compositional and physicochemical heterogeneity is a ubiquitous feature of cellular membranes on various length scales, from molecular assemblies to micrometric domains. Segregated lipid domains of increased local order, referred to as rafts, are believed to be prominent features in eukaryotic plasma membranes; however, their exact nature (i.e. size, lifetime, composition, homogeneity) in live cells remains difficult to define. Here we present evidence that both synthetic and natural plasma membranes assume a wide range of lipid packing states with varying levels of molecular order. These states may be adapted and specifically tuned by cells during active cellular processes, as we show for stimulated insulin secretion. Most importantly, these states regulate both the partitioning of molecules between coexisting domains and the bioactivity of their constituent molecules, which we demonstrate for the ligand binding activity of the glycosphingolipid receptor GM1. These results confirm the complexity and flexibility of lipid-mediated membrane organization and reveal mechanisms by which this flexibility could be functionalized by cells.