Internalization of membrane proteins involves their recruitment into plasma membrane clathrin-coated pits, with which they are thought to interact by binding to AP-2 adaptor protein complexes. To investigate the interactions of membrane proteins with coated pits at the cell surface, we applied image correlation spectroscopy to measure directly and quantitatively the clustering of influenza hemagglutinin (HA) protein mutants carrying specific cytoplasmic internalization signals. The HA system enables direct comparison between isolated internalization signals, because HA itself is excluded from coated pits. The studies presented here provide, for the first time, a direct quantitative measure for the degree of clustering of membrane proteins in coated pits at the cell surface. The degree of clustering depended on the strength of the internalization signal and on the integrity of the clathrin lattices and correlated with the internalization rates of the mutants. The clustering of the HA mutants fully correlated with their ability to co- precipitate α-adaptin from whole cells, the first such demonstration for a membrane protein that is not a member of the epidermal growth factor receptor family. Furthermore, both the clustering in coated pits and the co- precipitation with α-adaptin were dramatically reduced in the cold, suggesting that low temperature can interfere with the sorting of proteins into coated pits. In addition to the specific results reported here, the general applicability of the image correlation spectroscopy approach to study any process involving the clustering or oligomerization of membrane receptors at the cell surface is discussed.
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