ABSTRACT
Cell-cell contacts are fundamental to multicellular organisms and are subject to exquisite levels of control. The carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) can engage in both cis-homophilic (parallel) oligomerization and trans-homophilic (anti-parallel) binding. In this study, we establish that the CEACAM1 transmembrane domain has a propensity to form cis-dimers via the transmembrane-embedded (432)GXXXG(436) motif and that this basal state is overcome when activated calmodulin binds to the CEACAM1 cytoplasmic domain. Although mutation of the (432)GXXXG(436) motif reduced CEACAM1 oligomerization, it did not affect surface localization of the receptor or influence CEACAM1-dependent cellular invasion by the pathogenic Neisseria. The mutation did, however, have a striking effect on CEACAM1-dependent cellular aggregation, increasing both the kinetics of cell-cell association and the size of cellular aggregates formed. CEACAM1 association with tyrosine kinase c-Src and tyrosine phosphatases SHP-1 and SHP-2 was not affected by the (432)GXXXG(436) mutation, consistent with their association with the monomeric form of wild type CEACAM1. Collectively, our results establish that a dynamic oligomer-to-monomer shift in surface-expressed CEACAM1 facilitates trans-homophilic binding and downstream effector signaling.
Subject(s)
Antigens, CD/chemistry , Antigens, CD/metabolism , Cell Adhesion Molecules/chemistry , Cell Adhesion Molecules/metabolism , Protein Multimerization , Signal Transduction , Amino Acid Motifs/genetics , Amino Acid Sequence , Antigens, CD/genetics , Calcium/metabolism , Calmodulin/metabolism , Cell Adhesion , Cell Adhesion Molecules/genetics , Cell Membrane/metabolism , HeLa Cells , Humans , Immunoblotting , Microscopy, Confocal , Models, Molecular , Molecular Sequence Data , Mutation , Protein Binding , Protein Structure, Tertiary , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 6/metabolism , src-Family Kinases/metabolismABSTRACT
FGF21 stimulates FGFR1c activity in cells that co-express Klothoß (KLB); however, relatively little is known about the interaction of these receptors at the plasma membrane. We measured the dynamics and distribution of fluorescent protein-tagged KLB and FGFR1c in living cells using fluorescence recovery after photobleaching and number and brightness analysis. We confirmed that fluorescent protein-tagged KLB translocates to the plasma membrane and is active when co-expressed with FGFR1c. FGF21-induced signaling was enhanced in cells treated with lactose, a competitive inhibitor of the galectin lattice, suggesting that lattice-binding modulates KLB and/or FGFR1c activity. Fluorescence recovery after photobleaching analysis consistently revealed that lactose treatment increased KLB mobility at the plasma membrane, but did not affect the mobility of FGFR1c. The association of endogenous KLB with the galectin lattice was also confirmed by co-immunoprecipitation with galectin-3. KLB mobility increased when co-expressed with FGFR1c, suggesting that the two receptors form a heterocomplex independent of the galectin lattice. Number and brightness analysis revealed that KLB and FGFR1c behave as monomers and dimers at the plasma membrane, respectively. Co-expression resulted in monomeric expression of KLB and FGFR1c consistent with formation of a 1:1 heterocomplex. Subsequent addition of FGF21 induced FGFR1 dimerization without changing KLB aggregate size, suggesting formation of a 1:2 KLB-FGFR1c signaling complex. Overall, these data suggest that KLB and FGFR1 form a 1:1 heterocomplex independent of the galectin lattice that transitions to a 1:2 complex upon the addition of FGF21.
