| L-selectin is the central regulator of lymphocyte homing to lymph nodes. Its expression is subject to regulation by a membrane metalloprotease. Upon activation, cell surface L-selectin is proteolytically cleaved by the protease and its extracellular domain is shed. Shed L-selectin fragments exist at high levels in the sera of healthy humans and still retain ligand binding activity.; Previous studies demonstrated that lymphocytes constitutively shed surface L-selectin at a low basal level, and the activation of protein kinase C by PMA dramatically increases the shedding rate. The first part of the present study was designed to determine how the membrane protease recognizes and cleaves L-selectin. Mutagenesis analysis revealed that shedding of murine L-selectin has a relaxed sequence specificity for the cleavage site, but that a minimal length of the membrane-proximal region (MPR) is required for shedding. Interestingly, constitutive and PMA-stimulated shedding of L-selectin have different structural requirements. A proline substitution mutation in the MPR specifically blocks rapid PMA-induced shedding, but not slow basal shedding. Furthermore, the MPR of L-selectin can fully confer constitutive shedding upon a heterologous surface protein B7.2. The EGF domain is required for PMA-induced shedding of L-selectin and can also confer PMA-inducibility of shedding upon MPR-containing B7.2.; It has been hypothesized that L-selectin shedding is required during leukocyte transendothelial migration. The second part of the present study was designed to explore the physiological significance of rapid L-selectin shedding in vivo, especially in the transmigration of lymphocytes across HEV endothelium. Transgenic mice expressing only an L-selectin mutant which is resistant to both constitutive and PMA-induced shedding in vitro were generated. L-selectin shedding as measured after PMA-stimulation in vitro is not required for lymphocyte HEV-binding, transmigration across the HEV, or homing to lymph nodes. However, near-normal levels of soluble L-selectin were detected in the sera of the transgenic mice. The possibility that L-selectin shedding in vivo is mediated through a process distinct from that mimicked by the PMA-treatment in vitro was raised. |
