IDENTIFICATION OF A SIGNAL SEQUENCE NECESSARY FOR THE UNCONVENTIONAL SECRETION OF ENGRAILED HOMEOPROTEIN

Background: Engrailed-land Engrailed-2 are homeoproteins - transcripti on factors implicated in the morphogenesis of discrete structures. Eng railed proteins have a role in patterning the midbrain-hindbrain regio n and are expressed in the nuclei of rat embryo midbrain-hindbrain cel ls. We have previously found that both endogenous and exogenously expr essed Engrailed proteins also associate with membrane regions implicat ed in signal transduction and secretion. Within total membrane fractio ns, a small proportion of Engrailed - about 5% - is protected against proteinase K proteolysis, suggesting that Engrailed has access to a lu minal compartment. Together with our finding that homeodomains and hom eoproteins can be internalized by live cells, these observations sugge st that Engrailed might act as a polypeptidic messenger. In order to i nvestigate this possibility, we looked to see if Engrailed could be se creted. Results: Engrailed expressed in COS cells can be recovered in abutting primary neurons and this is dependent on a short sequence in its homeodomain distinct from 'classical' secretion signals. This sequ ence, which overlaps with the sequence necessary for Engrailed interna lization and which is highly conserved among homeoproteins, is the fir st example oi an 'unconventional' sequence necessary for secretion. Le ss than 5% of total intracellular Engrailed is secreted and there is a correlation between secretion and access to the membrane compartment where the protein is protected against proteinase K, Conclusions: Our results lend weight to the proposal that Engrailed, and possibly other homeoproteins, might act as intercellular polypeptidic messengers.