Signal transduction pathways as targets for therapeutics.

Meeting information: AAAS 2001 Annual Meeting and Science Innovation Exposition, San Francisco, California, February 15 through 20, 2001. Science's STKE sponsored a symposium at the AAAS Annual Meeting in February 2001. Five speakers addressed the signaling pathways that are modified in wide-ranging pathologies including inflammation, impotence, diabetes, obesity, and cancer. The molecular targets of signaling pathways included cell surface molecules, such as the G protein-coupled receptors (GPCRs) and receptor tyrosine kinases, and intracellular signaling components, such as phosphodiesterases (PDEs) and components of the small guanosine triphosphatase (GTPase) Ras signaling pathway. Analysis of the therapeutic strategies to impinge on these various pathways provides insight into both the potential of signaling pathways as relevant drug targets and the possible pitfalls that make complex signaling networks unpredictably difficult targets for such manipulation.

Utilization of the indirect lysosome targeting pathway by lysosome-associated membrane proteins (LAMPs) is influenced largely by the C-terminal residue of their GYXXphi targeting signals.

A systematic study was conducted on the requirements at the C-terminal position for the targeting of LAMPs to lysosomes, examining the hypothesis that a bulky hydrophobic residue is required. Mutations deleting or replacing the C-terminal valine with G, A, C, L, I, M, K, F, Y, or W were constructed in a reporter protein consisting of the lumenal/extracellular domain of avian LAMP-1 fused to the transmembrane and cytoplasmic domains of LAMP-2b. The steady-state distribution of each mutant form in mouse L-cells was assessed by quantitative antibody binding assays and immunofluorescence microscopy; efficiency of internalization from the plasma membrane and delivery to the lysosome were also estimated. It is found that (a) only C-terminal V, L, I, M, and F mediated efficient targeting to lysosomes, demonstrating the importance hydrophobicity and an optimal size of the C-terminal residue in targeting; (b) efficiency of lysosomal targeting generally correlated with efficiency of internalization; and (c) mutant forms that did not target well to lysosomes showed unique distributions in cells rather than simply default accumulation in the plasma membrane. Interactions of the targeting signals with adaptor subunits were measured using a yeast two-hybrid assay. The results are consistent with the hypothesis that trafficking of LAMP forms in cells through the indirect pathway is determined by the affinities of their targeting signals, predominantly for the mu2 and mu3 adaptors involved at plasma membrane and endosomal cellular sorting sites, respectively.

Different steady state subcellular distributions of the three splice variants of lysosome-associated membrane protein LAMP-2 are determined largely by the COOH-terminal amino acid residue.

The extensively glycosylated lysosome-associated membrane proteins (LAMP)-2a, b, and c are derived from a single gene by alternative splicing that produces proteins with differences in the transmembrane and cytosolic domains. The lysosomal targeting signals reside in the cytosolic domain of these proteins. LAMPs are not restricted to lysosomes but can also be found in endosomes and at the cell surface. We investigated the subcellular distribution of chimeras comprised of the lumenal domain of avian LAMP-1 and the alternatively spliced domains of avian LAMP-2. Chimeras with the LAMP-2c cytosolic domain showed predominantly lysosomal distribution, while higher levels of chimeras with the LAMP-2a or b cytosolic domain were present at the cell surface. The increase in cell surface expression was due to differences in the recognition of the targeting signals and not saturation of intracellular trafficking machinery. Site-directed mutagenesis defined the COOH-terminal residue of the cytosolic tail as critical in governing the distributions of LAMP-2a, b, and c between intracellular compartments and the cell surface.

Oligomerization of chicken acetylcholinesterase does not require intersubunit disulfide bonds.

Acetylcholinesterase (AChE) is secreted from muscle and nerve cells and associates as multimers through intermolecular covalent and noncovalent bonds. The amino acid sequence of the C-terminus is thought to play an important role in these interactions. We generated mutants in the C-terminus of the catalytic T-subunit of chicken AChE to determine the importance of this region to oligomerization and to the amphipathic character of the protein. Wild-type recombinant chicken AChE secreted from human embryonic kidney 293 cells was assembled into dimers and tetramers exclusively. Mutants lacking the C-terminal Cys764, the only cysteine involved in interchain disulfide bonds, showed lower but significant levels of the secreted dimeric and tetrameric forms. A truncated mutant, lacking the C-terminal 39 amino acids, exhibited a severe decrease in content of the multimeric forms, yet small amounts of the dimer were detectable. The amphipathic character was dependent on the state of oligomerization. When analyzed by sucrose gradients, the sedimentation of tetramers was not affected by detergent, but monomers and dimers sedimented more slowly in the presence of detergent. Most of the recombinant wild-type enzyme, shown to be dimeric and tetrameric by sedimentation analysis, was monomeric when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, indicating that much of the secreted oligomeric AChE was not disulfide bonded. These data suggest that disulfide bonding of Cys764 is not required for the catalytic subunit of chicken AChE to form oligomers and that regions outside of the C-terminus contribute to the hydrophobic interactions that are important for stabilizing the oligomeric forms.

The family of LAMP-2 proteins arises by alternative splicing from a single gene: characterization of the avian LAMP-2 gene and identification of mammalian homologs of LAMP-2b and LAMP-2c.

The two lysosome-associated membrane proteins, LAMP-1 and LAMP-2, are major integral membrane proteins of the lysosomes. They also occur in the plasma membrane, where they have been discovered independently as principal lactosaminoglycan-bearing glycoproteins and as tumor antigens. Avian LAMP-2 has recently been shown to be encoded by at least three transcripts resulting in variant transmembrane and cytoplasmic domains (Hatem et al., 1995). We report isolation and characterization of chicken genomic clones indicating that the three transcripts are the result of alternative splicing of a single LAMP-2 gene. Only a single LAMP-2, homologous to chicken LAMP-2a, has been described in mammals. To ascertain whether multiple forms of LAMP-2 also occur in mammals, we cloned cDNAs encoding LAMP-2 variants homologous to avian LAMP-2b and LAMP-2c from mouse brain cDNA libraries. Thus, the family of LAMP-2 proteins is conserved from bird to mammals and the diversity is generated by alternative splicing of a single LAMP-2 gene.

Multiple mRNAs encode the avian lysosomal membrane protein LAMP-2, resulting in alternative transmembrane and cytoplasmic domains.

Lysosomal membranes are enriched in extensively glycosylated transmembrane proteins, LAMP-1 and LAMP-2. LAMP-1 proteins have been characterized from several mammalian species and from chickens, but no non-mammalian homologues of LAMP-2 have been described, and no splice variants of either protein have been reported. Here we report the characterization of three cDNA clones encoding chicken LAMP-2. The nucleotide sequences of the cDNAs diverge at their 3' ends within the open reading frame, resulting in sequences that code for three different transmembrane and cytoplasmic domains. Southern analysis suggests that a single gene encodes the common region of chicken LAMP-2. The position of the divergence and the identity of the common sequence are consistent with alternative splicing of 3' exons. Analysis of the mRNAs present in adult chicken tissues suggests tissue-specific expression of the three chicken LAMP-2 variants, with LAMP-2b expressed primarily in the brain. The cytoplasmic domain of LAMP-type proteins contains the targeting signal for directing these molecules to the lysosome. Using chimeras consisting of the lumenal domain of chicken LEP100 (a LAMP-1) and the transmembrane and cytoplasmic domains of the LAMP-2 variants, we demonstrate in transfected mouse L cells that all three LAMP-2 carboxyl-terminal regions are capable of targeting the chimeric proteins to lysosomes. Levels of expression, subcellular distribution, and glycosylation of the LAMP proteins have all been shown to change with differentiation in mammalian cells and to be correlated with metastatic potential in certain tumor cell lines. Alternative splicing of the LAMP-2 transcript may play a role in these changes.

Cloning and analysis of chicken acetylcholinesterase transcripts from muscle and brain.

We have isolated cDNA clones from an embryonic chicken muscle cDNA library which encodes the complete catalytic T subunit of acetylcholinesterase. The deduced polypeptide comprises 767 amino acids, shows approximately 60% homology to acetylcholinesterases from other vertebrates and contains a 155 amino acid sequence inserted into the middle of the peptide which is unique to the chicken enzyme. Northern blots of embryonic chicken muscle and adult brain show three transcripts approximately 4.5, 5.5, and 6.0 kb hybridizing to a cDNA fragment of AChE. The 6.0 kb transcript is expressed transiently in embryonic muscle and is a major transcript in adult brain.