Tracking the Cartoon mouse phenotype: Hemopexin domain-dependent regulation of MT1-MMP pericellular collagenolytic activity.

Following ENU mutagenesis, a phenodeviant line was generated, termed the "Cartoon mouse," that exhibits profound defects in growth and development. Cartoon mice harbor a single S466P point mutation in the MT1-MMP hemopexin domain, a 200-amino acid segment that is thought to play a critical role in regulating MT1-MMP collagenolytic activity. Herein, we demonstrate that the MT1-MMP mutation replicates the phenotypic status of -null animals as well as the functional characteristics of MT1-MMP cells. However, rather than a loss-of-function mutation acquired as a consequence of defects in MT1-MMP proteolytic activity, the S466P substitution generates a misfolded, temperature-sensitive mutant that is abnormally retained in the endoplasmic reticulum (ER). By contrast, the WT hemopexin domain does not play a required role in regulating MT1-MMP trafficking, as a hemopexin domain-deletion mutant is successfully mobilized to the cell surface and displays nearly normal collagenolytic activity. Alternatively, when MT1-MMP-expressing cells are cultured at a permissive temperature of 25 °C that depresses misfolding, the mutant successfully traffics from the ER to the -Golgi network (ER → -Golgi network), where it undergoes processing to its mature form, mobilizes to the cell surface, and expresses type I collagenolytic activity. Together, these analyses define the Cartoon mouse as an unexpected gain-of- function mutation, wherein the temperature-sensitive mutant phenocopies MT1-MMP mice as a consequence of eliciting a specific ER → -Golgi network trafficking defect.