Detecting cell membrane protein endocytosis

A method for determining endocytic changes of a cell membrane receptor in mammalian cells as a result of a change of environment of said cells where said cells are suspended in a medium and genetically modified to express a labeled cell membrane receptor, said label being a polypeptide and a first member of a specific binding pair, and external to said cell membrane, and said method including as a reagent a complementary antibody to said polypeptide joined to a β-galactosidase member of an enzyme fragment complementation pair which, when complexed to the complementary enzyme fragment, forms an active β-galactosidase, said method comprising:

• adding said reagent to said cells suspended in a medium and incubating for sufficient time for said reagent to bind to said label of said cell membrane receptor;
• adding a test compound to said cells to determine the effect of said test compound on endocytosis of said cell membrane receptor wherein other than endocytosed reagent remains bound to said labeled cell membrane receptor and said β-galactosidase member of said reagent remains external to said cell membrane;
• degrading external β-galactosidase member of said enzyme fragment complementation pair with at least one protease;
• deactivating said at least one protease;
• lysing said cells by osmotic shock;
• adding said complementary enzyme fragment and a substrate;
• and
• determining the amount of product produced from said substrate;
• wherein the amount of product is related to the amount of endocytosis of said cell membrane receptor.

A kit comprising:

• (1) (a) a mammalian cell comprising an expression construct expressing a cell membrane receptor fused at its external end to a polypeptide label or (b) an expression construct expressing a cell membrane receptor fused at its external end to a polypeptide label;
• (2) antibody to said polypeptide label joined to a first β-galactosidase fragment;
• and
• (3) a complementary second β-galactosidase fragment that when complexed with said first fragment forms an active enzyme.