In the cell, as in vitro, the final conformation of a protein is determined by its amino-acid sequence. But whereas some isolated proteins can be denatured and refolded in vitro in the absence of other macromolecular cellular components, folding and assembly of polypeptides in vivo involves other proteins, many of which belong to families that have been highly conserved during evolution.

Protein folding in the cell.

Proteins that contain the Arg-Gly-Asp (RGD) attachment site, together with the integrins that serve as receptors for them, constitute a major recognition system for cell adhesion. The RGD sequence is the cell attachment site of a large number of adhesive extracellular matrix, blood, and cell surface proteins, and nearly half of the over 20 known integrins recognize this sequence in their adhesion protein ligands. Some other integrins bind to related sequences in their ligands. The integrin-binding activity of adhesion proteins can be reproduced by short synthetic peptides containing the RGD sequence. Such peptides promote cell adhesion when insolubilized onto a surface, and inhibit it when presented to cells in solution. Reagents that bind selectively to only one or a few of the RGD-directed integrins can be designed by cyclizing peptides with selected sequences around the RGD and by synthesizing RGD mimics. As the integrin-mediated cell attachment influences and regulates cell migration, growth, differentiation, and apoptosis, the RGD peptides and mimics can be used to probe integrin functions in various biological systems. Drug design based on the RGD structure may provide new treatments for diseases such as thrombosis, osteoporosis, and cancer.

RGD and other recognition sequences for integrins.

Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP.

Cyclooxygenase (COX), first purified in 1976 and cloned in 1988, is the key enzyme in the synthesis of prostaglandins (PGs) from arachidonic acid. In 1991, several laboratories identified a product from a second gene with COX activity and called it COX-2. However, COX-2 was inducible, and the inducing stimuli included pro-inflammatory cytokines and growth factors, implying a role for COX-2 in both inflammation and control of cell growth. The two isoforms of COX are almost identical in structure but have important differences in substrate and inhibitor selectivity and in their intracellular locations. Protective PGs, which preserve the integrity of the stomach lining and maintain normal renal function in a compromised kidney, are synthesized by COX-1. In addition to the induction of COX-2 in inflammatory lesions, it is present constitutively in the brain and spinal cord, where it may be involved in nerve transmission, particularly that for pain and fever. PGs made by COX-2 are also important in ovulation and in the birth process. The discovery of COX-2 has made possible the design of drugs that reduce inflammation without removing the protective PGs in the stomach and kidney made by COX-1. These highly selective COX-2 inhibitors may not only be anti-inflammatory but may also be active in colon cancer and Alzheimer’s disease.

Cyclooxygenases 1 and 2

Neurotechnique Imaging Neuronal Subsets in Transgenic Mice Expressing Multiple Spectral Variants of GFP

Defective Neuromuscular Synaptogenesis in Agrin-Deficient Mutant Mice

A striking example of topographic specificity in synapse formation is the preferential reinnervation of original synaptic sites on denervated muscle fibres by regenerating motor axons. This specificity is mediated by the basal lamina of the synaptic cleft. A glycoprotein, s-laminin, has now been iden-tified that is selectively associated with synaptic basal lamina and is recognized by motoneurons. Molecular cloning reveals that s-laminin is a novel homologue of laminin, a potent promoter of neurite outgrowth.

A laminin-like adhesive protein concentrated in the synaptic cleft of the neuromuscular junction

OF numerous synaptic components that have been identified, perhaps the best-studied are the nicotinic acetylcholine receptors (AChRs) of the vertebrate neuromuscular junction1. AChRs are diffusely distributed on embryonic myotubes, but become highly concentrated (~ 10,000 µm-2) in the postsynaptic membrane as development proceeds. At least two distinct processes contribute to this accumulation. One is local synthesis: subsynaptic muscle nuclei transcribe AChR subunit genes at higher rates than extra-synaptic nuclei, so AChR messenger RNA is concentrated near synaptic sites2,3. Second, once AChRs have been inserted in the membrane, they form high-density clusters by tethering to a sub-synaptic cytoskeletal complex. A key component of this complex is rapsyn, a peripheral membrane protein of relative molecular mass 43K (refs 4, 5), which is precisely colocalized with AChRs at synaptic sites from the earliest stages of neuromuscular synaptogenesis6. In heterologous systems, expression of recombi-nant rapsyn leads to clustering of diffusely distributed AChRs, suggesting that rapsyn may control formation of clusters7,8. To assess the role of rapsyn in vivo, we generated and characterized mutant mice with a targeted disruption of the Rapsn gene. We report that rapsyn is essential for the formation of AChR clusters, but that synapse-specific transcription of AChR subunit genes can proceed in its absence.

Failure of postsynaptic specialization to develop at neuromuscular junctions of rapsyn-deficient mice

SYNAPSE formation requires a complex interchange of information between the pre- and postsynaptic partners. At the skeletal neuro-muscular junction, some of this information is contained in the basal lamina (BL), which runs through the synaptic cleft between the motor nerve terminal and the muscle fibre. During regeneration following injury, components of synaptic BL can trigger several features of postsynaptic differentiation in the absence of the nerve terminal, and of presynaptic differentiation in the absence of the muscle fibre1–3. One nerve-derived component of synaptic BL, agrin, is known to affect postsynaptic differentiation3, but no muscle-derived components have yet been shown to influence motor nerve terminals. A candidate for such a role is s-laminin (also called laminin β2), a homologue of the Bl (β1) chain of the widely distributed BL glycoprotein, laminin30. s-laminin is synthesized by muscle cells5 and concentrated in synaptic BL4. In vitro, recombinant s-laminin fragments are selectively adhesive for motor neuron-like cells, inhibit neurite outgrowth promoted by other matrix molecules, and act as a 'stop signal' for growing neurites6,7. By generating and characterizing mice with a targeted mutation of the S-laminin gene, we show here that s-laminin regulates formation of motor nerve terminals.

John P. Merlie

Papers

Defective Neuromuscular Synaptogenesis in Agrin-Deficient Mutant Mice

A laminin-like adhesive protein concentrated in the synaptic cleft of the neuromuscular junction

Failure of postsynaptic specialization to develop at neuromuscular junctions of rapsyn-deficient mice

Aberrant differentiation of neuromuscular junctions in mice lacking s-laminin/laminin β2

Isolation and characterization of the complementary DNA for sheep seminal vesicle prostaglandin endoperoxide synthase (cyclooxygenase).