Agrin isoforms with distinct amino termini: Differential expression, localization, and function

The proteoglycan agrin is required for postsynaptic differentiation at the skeletal neuromuscular junction, but is also associated with basal laminae in numerous other tissues, and with the surfaces of some neurons. Little is known about its roles at sites other than the neuromuscular junction, or a bout how its expression and subcellular localization are regulated in any t issue. Here we demonstrate that the murine agrin gene generates two protein s with different NH2 termini, and present evidence that these isoforms diff er in subcellular localization. tissue distribution, and function. The two isoforms share similar to 1,900 amino acids (aa) of common sequence followi ng unique NH2 termini of 49 or 150 aa; we therefore call them short NH2-ter minal (SN) and long NH2-terminal (LN) isoforms. In the mouse genome, LN-spe cific exons are upstream of an SN-specific exon. which is in turn upstream of common exons, LN-agrin is expressed in both neural and nonneural tissues . In spinal cord it is expressed in discrete subsets of cells, including mo toneurons. In contrast, SN-agrin is selectively expressed in the nervous sy stem but is widely distributed in many neuronal cell types. Both isoforms a re externalized from cells but LN-agrin assembles into basal laminae wherea s SN-agrin remains cell associated. Differential expression of the two isof orms appears to be transcriptionally regulated, whereas the unique SN and L N sequences direct their distinct subcellular localizations. Insertion of a "gene trap" construct into the mouse genome between the LN and SN exons ab olished expression of LN-agrin with no detectable effect on expression leve ls of SN-agrin or on SN-agrin bioactivity in vitro. Agrin protein was absen t from all basal laminae in mice lacking LN-agrin transcripts. The formatio n of the neuromuscular junctions was as drastically impaired in these mutan ts as in mice lacking all forms of agrin,Thus, basal lamina-associated LN-a grin is required for neuromuscular synaptogenesis, whereas cell-associated SN-agrin may play distinct roles in the central nervous system.