Lay summary
White blood cells, also called leukocytes, are the immune cells of the body. Examples of leukocytes
are T lymphocytes and neutrophils. T lymphocytes are activated upon viral infection. In order to be
effective, T lymphocytes migrate to specific cellular compartments called lymph nodes where they
interact with B lymphocytes. Such interaction is crucial for the production of antibodies against
invading viruses. Neutrophils migrate into infected tissues where they capture and destroy bacteria
and fungi. In order for T lymphocytes and neutrophils to migrate into infected tissues or specialized
compartments, they must leave the blood circulation by crossing the blood vessel. To this end, they
need to adhere to cells which cover the inside of the blood vessels called endothelial cells. Such
adhesion requires receptors expressed on the membrane surface of leukocytes (adhesion receptors)
which bind specific molecules (called ligands) present on the cell surface of endothelial cells. Once
bound to endothelial cells, leukocytes can pass through junctions between endothelial cells and reach
tissues. One of the key adhesion receptor is L-selectin. The aim of the project was to understand how
this receptor is activated during the immune response to allow leukocytes to adhere to endothelial
cells and migrate outside the blood circulation. To do this, we aimed at identifying proteins
interacting with L-selectin with the idea that these interacting proteins could control the ability of Lselectin to exhibit adhesive capacities. We found a novel protein called AP-1 mu1A that binds to Lselectin. AP-1 mu1A is a protein involved in the transport of receptors. This finding suggests that the
adhesive capacity of L-selectin may be controlled by the regulated transport of this receptor to the
surface of leukocyte membrane. Furthermore, we identified the region in L-selectin involved in the
interaction with AP-1 mu1A. To prove that binding of AP-1 mu1A with L-selectin regulates the
migration of leukocytes in animals, we designed a mouse in which the region of L-selectin gene
involved in the interaction with AP-1 mu1A was mutated. With this model, we will be able in the
future to understand the role played by AP-1 mu1A in the regulation of L-selectin adhesive functions
in living mice and the contribution of the AP-1 mu1A/L-selectin interaction for the immune response.
Scientific summary
By using L-selectin tail peptide pull-down assays combined with sophisticated mass spectrometrybased proteomics, we identified the AP-1 mu1A adaptin (AP1m1 gene) as a novel L-selectin tail
interactor. AP-1 mu1A is one of the four subunits of the AP-1 complex. AP-1 mu1A interacts with
sorting motifs of transmembrane proteins (called cargo proteins) to incorporate them into clathrincoated vesicles. AP-1 mediates bi-directional transport of proteins between the trans-Golgi Network
(TGN) and early endosomes via clathrin-coated-vesicles. We confirmed this finding by showing that
full length mu1A and the C-terminal cargo binding part of mu1A (C-mu1A) expressed as GST fusion
proteins bind the L-selectin peptide sequence. Control experiments showed that GST alone as well as
the N-terminal part of mu1A, which is bound by the β subunit of AP-1, did not interact with the Lselectin tail peptide.
Based on the fact that proteins with clusters of positive charges interact with mu1A, we investigated
whether the dibasic clusters of the RRLKKGKK sequence of L-selectin tail were involved in the
interaction with mu1A. This was the case. Indeed, changing arginine (R) or lysine (K) residues to
alanine within any of the dibasic motifs totally prevented the binding of full length GST-mu1A or
GST-C-mu1A to the L-selectin tail. Information gained from these studies will be used to model the
interaction between the novel non canonical sequence of L-selectin tail and mu1A.
Serine 364 of L-selectin tail is phosphorylated in activated leukocytes. It is thought that such covalent
modification modulates the interaction between L-selectin tail and binding partners and thereby
adhesive functions of the receptor. Next, we investigated whether serine phosphorylation of Lselectin tail influences the binding of mu1A. We showed that GST-mu1A or GST-C-mu1A do not
bind the L-selectin tail peptide in which serine 364 is phosphorylated. A similar result was found
when serine 364 was replaced by aspartic acid, an acidic amino-acid to imitate a constitutively
phosphorylated residue. Our results suggest that L-selectin tail is phosphorylated in endosomes or
TGN by a clathrin-associated kinase. Such phosphorylation may regulate L-selectin accumulation in
endosomes and localization of clustered receptor on the tips of microvilli on the plasma membrane.
To unambiguously prove this, we designed a mouse harbouring a non-phosphorylatable serine-toalanine knock-In (KI) substitution at position serine 364 of the L-selectin tail (KI S364A mouse). The
analysis of L-selectin trafficking and localization as well as L-selectin-dependent rolling in
leukocytes from the KI S364A mouse will reveal the crucial role played by the dynamic
phosphorylation of L-selectin tail for in vivo leukocyte migration.
Significance of the research project and potential impact: There is a lack of knowledge on the
mechanisms by which L-selectin regulates leukocyte adhesion and migration. Our discovery of the
AP-1 mu1A-adaptin as a novel interactor of the L-selectin tail is of significant importance. First, it
suggests that surface expression of L-selectin is not only regulated by proteolytic cleavage in the
plasma membrane, but also by the regulation of its intracellular trafficking. Thus, L-selectin
trafficking may control replenishment of the L-selectin pool on the plasma membrane surface upon
proteolytic cleavage. Second, regulated L-selectin trafficking may explain how L-selectin clusters are
formed on the tips of microvilli to allow leukocyte rolling along the endothelium. Third, L-selectin
trafficking may be dynamically regulated through phosphorylation of L-selectin tail in endosomes
and/or the TGN. Our discovery will have an impact on the comprehension of L-selectin function
Conclusions and socio-economic impacts of the project: The main objectives of the proposal were
reached. The Fellow identified a novel protein interacting with L-selectin cytoplasmic tail and
designed a L-selectin KI S364A mouse. Advanced skills in Molecular Biology, Biochemistry and
Proteomics-based mass spectrometry were transferred to Queen’s University of Belfast through
extensive training of the Fellow at the Max Planck Institute of Biochemistry. Thus, new and modern
technology platforms can be developed in a UK-based university. The L-selectin KI S364A mouse
model will be transferred to Queen’s University of Belfast where this promising study will be further
developed with the goal of generating publications of excellent scientific standard. The Fellow is
currently seeking for funding from UK-based research grant bodies (BBSRC and Wellcome Trust) to
pursue this work. Clearly, the preliminary data generated at the Max Planck Institute of Biochemistry
including the design of the L-selectin KI S364A mouse model will be crucial for the success of grant
applications. This means that new post-Doctoral Fellows and Ph.D students will be employed to
Queen’s University Belfast to carry out this work thus bringing economic wealth to Northern Ireland.
The research is fundamental and will be relevant to the Academic society, but on the long term, this
knowledge could be exploited in Research and Development to design novel anti-inflammatory
compounds directed against Kinases which phosphorylate L-selectin tail or proteins interacting with
L-selectin tail.