ENDOCYTIC CLATHRIN-COATED PIT FORMATION IS INDEPENDENT OF RECEPTOR INTERNALIZATION SIGNAL LEVELS

The mechanisms responsible for coated pit formation in cells remain un known, but indirect evidence has argued both for and against a critica l role of receptor cytoplasmic domains in the process. If the endocyti c motifs of receptors are responsible for recruiting AP2 to the plasma membrane, thereby driving coated pit formation, then the level of con stitutively internalized receptors at the membrane would be expected t o govern the steady-state level of coated pits in cells. Here we direc tly test this hypothesis for broad classes of receptors containing thr ee distinct constitutive internalization signals. Chimeric proteins co nsisting of an integral membrane reporter protein (Tac) coupled to cyt oplasmic domains bearing tyrosine-, di-leucine-, or acidic cluster/cas ein kinase II-based internalization signals were overexpressed to leve ls that saturated the internalization pathway. Quantitative confocal i mmunofluorescence microscopy indicated that the number of plasma membr ane clathrin-coated pits and the concentration of their structural com ponents were invariant when comparing cells expressing saturating leve ls of the chimeric receptors to nonexpressing cells or to cells expres sing only the Tac reporter lacking cytoplasmic internalization signals . Biochemical analysis showed that the distribution of coat proteins b etween assembled coated pits and soluble pools was also not altered by receptor overexpression. Finally, the cellular localizations of AP2 a nd AP1 were similarly unaffected. These results provide a clear indica tion that receptor endocytic signals do not determine coated pit level s by directly recruiting AP2 molecules. Rather, the findings support a model in which coated pit formation proceeds through recruitment and activation of AP2, likely through a limited number of regulated dockin g sites that act independently of endocytic signals.