Novel isoform of insulin receptor substrate p53/p58 is generated by alternative splicing in the CRIB/SH3-binding region

Insulin receptor substrate p53/p58 (IRSp53) is involved in cytoskeletal dynamics and is a candidate disease sensor in polyglutamine expansion neurodegeneration. It is widely expressed throughout the body, but its levels are dramatically elevated in forebrain regions. IRSp53 functions as a signal transducing adaptor between activated Rho family GTPases and their effectors. There are four known alternatively spliced isoforms of IRSp53 that vary by the identity of the 3'-terminal exon. We report here that there is a fifth alternatively spliced isoform, IRSp53-B, which lacks 40 amino acids abutting the CRIB/SH3 (Cdc42/Rac-interactive binding/Src homology 3)-binding site. We speculate that the novel form has an altered function related to the mechanism of autoinactivation. IRSp53-B has an odd history in the mammalian lineage, which may complicate the use of rodent models to study cytoskeletal reorganization.     

Functional conservation of subfamilies of putative UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases in Drosophila, Caenorhabditis elegans, and mammals. One subfamily composed of l(2)35Aa is essential in Drosophila

The completed fruit fly genome was found to contain up to 15 putative UDP-N-acetyl-alpha-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase (GalNAc-transferase) genes. Phylogenetic analysis of the putative catalytic domains of the large GalNAc-transferase enzyme families of Drosophila melanogaster (13 available), Caenorhabditis elegans (9 genes), and mammals (12 genes) indicated that distinct subfamilies of orthologous genes are conserved in each species. In support of this hypothesis, we provide evidence that distinctive functional properties of Drosophila and human GalNAc-transferase isoforms were exhibited by evolutionarily conserved members of two subfamilies (dGalNAc-T1 (l(2)35Aa) and GalNAc-T11; dGalNAc-T2 (CG6394) and GalNAc-T7). dGalNAc-T1 and novel human GalNAc-T11 were shown to encode functional GalNAc-transferases with the same polypeptide acceptor substrate specificity, and dGalNAc-T2 was shown to encode a GalNAc-transferase with similar GalNAc glycopeptide substrate specificity as GalNAc-T7. Previous data suggested that the putative GalNAc-transferase encoded by l(2)35Aa had a lethal phenotype (Flores, C., and Engels, W. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 2964-2969), and this was substantiated by sequencing of three lethal alleles l(2)35Aa(HG8), l(2)35Aa(SF12), and l(2)35Aa(SF32). The finding that subfamilies of GalNAc-transferases with distinct catalytic functions are evolutionarily conserved stresses that GalNAc-transferase isoforms may serve unique biological functions rather than providing functional redundancy, and this is further supported by the lethal phenotype of l(2)35Aa.     

TAPAS-1, a novel microdomain within the unique N-terminal region of the PDE4A1 cAMP-specific phosphodiesterase that allows rapid, Ca2+-triggered membrane association with selectivity for interaction with phosphatidic acid

Here we identify an 11-residue helical module in the unique N-terminal region of the cyclic AMP-specific phosphodiesterase PDE4A1 that determines association with phospholipid bilayers and shows a profound selectivity for interaction with phosphatidic acid (PA). This module contains a core bilayer insertion unit that is formed by two tryptophan residues, Trp(19) and Trp(20), whose orientation is optimized for bilayer insertion by the Leu(16):Val(17) pairing. Ca(2+), at submicromolar levels, interacts with Asp(21) in this module and serves to gate bilayer insertion, which is completed within 10 ms. Selectivity for interaction with PA is suggested to be achieved primarily through the formation of a charge network of the form (Asp(21-):Ca(2+):PA(2-):Lys(24+)) with overall neutrality at the bilayer surface. This novel phospholipid-binding domain, which we call TAPAS-1 (tryptophan anchoring phosphatidic acid selective-binding domain 1), is here identified as being responsible for membrane association of the PDE4A1 cAMP-specific phosphodiesterase. TAPAS-1 may not only serve as a paradigm for other PA-binding domains but also aid in detecting related phospholipid-binding domains and in generating simple chimeras for conferring membrane association and intracellular targeting on defined proteins.     

Trypsin-sensitive and lipid-containing sites of the macrophage extracellular matrix bind apolipoprotein A-I and participate in ABCA1-dependent cholesterol efflux

A unique property of the extracellular matrix of J774 and THP-1 cells has been identified, which contributes to the ability of these cells to promote cholesterol efflux. We demonstrate high level apolipoprotein (apo) A-I binding to macrophage cells (THP-1 and J774) and to their extracellular matrix (ECM). However, high level apoA-I binding is not observed on fibroblasts, HepG2 cells, or U937 cells (a macrophage cell line that does not efflux cholesterol to apoA-I or bind apoA-I on their respective ECM). Binding to the ECM of THP-1 or J774 macrophages depends on the presence of apoA-I C-terminal helices and is markedly reduced with a mutant lacking residues 187-243 (apoA-I Delta(187-243)), suggesting that the hydrophobic C terminus forms a hydrophobic interaction with the ECM. ApoA-I binding is lost upon trypsin treatment or with Triton X-100, a preparation method that de-lipidates the ECM. However, binding is recovered with re-lipidation, and is preserved with ECM prepared using cytochalasin B, which conserves the endogenous phospholipid levels of the ECM. We also demonstrate that specific cholesterol efflux to apoA-I is much reduced in cells released from their native ECM, but fully restored when ECM-depleted cells are added back to ECM in the presence of apoA-I. The apoA-I-mediated efflux is deficient in plated or suspension U937 macrophages, but is restored to high levels when the suspension U937 cells are reconstituted with the ECM of J774 cells. The ECM-dependent activity was much reduced in the presence of glyburide, indicating participation of ABCA1 (ATP-binding cassette transporter 1) in the efflux mechanism. These studies establish a novel binding site for apoA-I on the macrophage ECM that may function together with ABCA1 in promoting cholesterol efflux.     

Sutural loosening and skeletal flexibility during growth: determination of drop-like shapes in sea urchins

The shape of sea urchins may be determined mechanically by patterns of force analogous to those that determine the shape of a water droplet. This mechanical analogy implies skeletal flexibility at the time of growth. Although comprised of many rigid calcite plates, sutural collagenous ligaments could confer such flexibility if the sutures between plates loosened and acted as joints at the time of growth. We present experimental evidence of such flexibility associated with weight gain and growth. Over 13-, 4-, and 2-week periods, fed urchins (Strongylocentrotus droebachiensis) gained weight and developed looser sutures than unfed urchins that maintained or lost weight. Further, skeletons of fed urchins force-relaxed more than did those of unfed urchins and urchins with loose sutures force-relaxed more than those with tight sutures. Urchins (Strongylocentrotus franciscanus) fed for two and a half weeks, gained weight, also had looser skeletons and deposited calcite at sutural margins, whereas unfed ones did not. In field populations of S. droebachiensis the percentage having loose sutures varied with urchin diameter and reflected their size-specific growth rate. The association between feeding, weight gain, calcite deposition, force relaxation and sutural looseness supports the hypothesis that urchins deform flexibly while growing, thus determining their drop-like shapes.     

Complements to the description of Tityus dedoslargos (Scorpiones: Buthidae)

The female of the Costarican scorpion Tityus dedoslargos Francke and Stockwell, 1987 is described for the first time and new data on its morphological variation and natural history are given. The female differs from the male mainly in having an enlarged basal median lamellae on the pectines, and lacking modified pedipalps. The species type locality relocated. This species is recorded from only two Costarican provinces: San Jose and Puntarenas.     

Centruroides Schmidti (scorpiones: Buthidae) in Costa Rica

Centruroides schmidti Sissom, 1995 is the new identification designate of the scorpions previously known as C. thorelli (Kraepelin, 1891) in Northwest Costa Rica. The geographical distribution of the species in this country is established on the basis of new collected material. Its distribution includes the Pacific slope of the Guanacaste volcanic range, and the Pacific and Atlantic slopes of the Tilarán volcanic range between 50 and 775 m elevation above sea level, within the Costa Rican Guanacaste and Alajuela provinces.