Agrin is a basal lamina molecule that directs key events in postsynaptic differentiation, most notably the aggregation of acetylcholine receptors (AChRs) on the muscle cell surface. Agrin's AChR clustering activity is regulated by alternative mRNA splicing. Agrin splice forms having inserts at two sites (y and z) in the C-terminal region are highly active, but isoforms lacking these inserts are weakly active. The biochemical consequences of this alternative splicing are unknown. Here, the binding of four recombinant agrin isoforms to heparin, to a-dystroglycan (a component of an agrin receptor), and to myoblasts was tested. The presence of a four-amino acid insert at they site is necessary and sufficient to confer heparin binding ability to agrin. Moreover, the binding of agrin to a-dystroglycan is inhibited by heparin when this insert is present. Agrin binding to the cell surface showed analogous properties: heparin inhibits the binding of only those agrin isoforms containing this four-amino acid insert. The results show that alternative splicing of agrin regulates its binding to heparin and suggest that agrin's interaction with ca-dystroglycan may be modulated by cell surface glycosaminoglycans in an isoform-dependent manner.
The distribution of muscarinic cholinergic binding sites in the striatum was studied in relation to the locations of other neurochemical markers in the developing rat, cat, ferret, and human. In addition, patterns of striatal muscarinic binding were studied in the adult cat. Receptor binding autoradiography was carried out with tritiated propylbenzilylcholine mustard [( 3H]-PrBCM), an irreversible muscarinic antagonist, and subsequent serial section analyses involved comparisons among patterns of muscarinic binding, catecholamine histofluorescence, acetylcholinesterase (AChE) staining, Nissl staining, and cell labeling with [3H]-thymidine. Muscarinic binding in the immature striatum was characterized by local patchiness as well as regional density gradients in all species, with the most complex patterns appearing in the human. Patches of dense muscarinic binding were shown to lie in register with fluorescent dopamine islands (rat, cat, ferret), with AChE-positive patches (all species), and with clusters of neurons pulse-labeled by exposure to [3H]-thymidine on embryonic day 27 (ferret). At the developmental stages examined, the [3H]-PrBCM-positive patches were roughly aligned with regions of weak Nissl staining (cat, human). Striatal [3H]-PrBCM binding in the adult cat was dense, and though it usually appeared nearly homogeneous, in some sections patches of elevated binding were present. These had as counterparts, in neighboring sections, AChE-poor striosomes. We conclude that during development muscarinic cholinergic function is compartmentalized in the striatum in association with dopamine-containing afferents, and that this compartmentalization may persist to some degree in the adult.
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