Intramembrane Proteolysis of Astrotactins

Astrotactins are vertebrate-specific membrane proteins implicated in neuron-glia interactions during central nervous system development and in hair follicle polarity during skin development. By studying epitope-tagged derivatives of mouse astrotactin-2 (Astn2) produced in transfected cells, we determined that the amino and carboxyl termini reside in the extracellular space and are initially linked by two transmembrane segments and a single cytoplasmic domain. We further show that Astn2 undergoes proteolytic cleavage in the second transmembrane domain (TM2) and that a disulfide bond holds the resulting two fragments together. Recombinant Astn1 also undergoes TM2 cleavage, as does Astn2 isolated from mouse cerebellum. Astn2 intramembrane proteolysis is insensitive to replacement of TM2 by the transmembrane domain of CD74 or by 21 alanines. However, replacement of TM2 by the transmembrane domain of CD4, the asialoglycoprotein receptor, or the transferrin receptor eliminates intramembrane proteolysis, as does leucine substitution of residues that overlap or are immediately upstream of the cleavage site. Replacement of the transmembrane domain of CD74 or the asialoglycoprotein receptor with Astn2 TM2 leads to the appearance of a carboxyl-terminal fragment consistent with intramembrane proteolysis. These experiments define a highly unusual transmembrane topology for the astrotactins, reveal intramembrane proteolysis as a feature of astrotactin maturation, and constrain the substrate sequences that are permissive for cleavage of one type 2 transmembrane segment.