Internalization-competent influenza hemagglutinin mutants form complexes with clathrin-deficient multivalent AP-2 oligomers in live cells

Most membrane proteins are endocytosed through clathrin-coated pits via AP- 2 adaptor complexes. However, little is known about the interaction of inte rnalization signals with AP-2 in live cells in the absence of clathrin latt ices. To investigate this issue, we employed cells cotransfected with pairs of antigenically distinct influenza hemagglutinin (HA) mutants containing different internalization signals of the YXXZ family. To enable studies on the possible association of the naturally trimeric HAs into higher order co mplexes via binding to AP-2, we exploited the inability of HAs from differe nt influenza strains to form mutual trimers. Thus, we coexpressed HA pairs from different strains (Japan and X:31) bearing similar cytoplasmic tails m utated to include internalization signals. Using antibody-mediated immunofl uorescence co-patching on live cells, we demonstrate that internalization-c ompetent HA mutants form higher order complexes and that this clustering de pends on the strength of the internalization signal. The clustering persist ed in cells treated with hypertonic medium to disperse the clathrin lattice s, as validated by co-immunoprecipitation experiments. The clustering of HA s bearing strong internalization signals appears to be mediated via binding to AP-2, as indicated by (i) the coprecipitation of alpha -adaptin with th ese HAs, even in hypertonically treated cells; (ii) the co-localization (af ter hypertonic treatment) of AP-2 with antibody-mediated patches of these m utants; and (iii) the dispersal of the higher order HA complexes following chlorpromazine treatment, which removes AP-2 from the plasma membrane. Thes e results suggest that even in the absence of clathrin lattices, AP-2 exist s in multivalent complexes capable of simultaneously binding several intern alization signals from the same family.