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Vol. 9, Issue 5, 1177-1194, May 1998
and
*Kimmel Cancer Institute and the Departments of Microbiology and
Immunology and of Biochemistry and Molecular Pharmacology, Thomas
Jefferson University, Philadelphia, Pennsylvania 19107; and
The mechanisms responsible for coated pit formation in cells remain
unknown, but indirect evidence has argued both for and against a
critical role of receptor cytoplasmic domains in the process. If the
endocytic motifs of receptors are responsible for recruiting AP2 to the
plasma membrane, thereby driving coated pit formation, then the level
of constitutively internalized receptors at the membrane would be
expected to govern the steady-state level of coated pits in cells. Here
we directly test this hypothesis for broad classes of receptors
containing three distinct constitutive internalization signals.
Chimeric proteins consisting of an integral membrane reporter protein
(Tac) coupled to cytoplasmic domains bearing tyrosine-, di-leucine-, or
acidic cluster/casein kinase II-based internalization signals were
overexpressed to levels that saturated the internalization pathway.
Quantitative confocal immunofluorescence microscopy indicated that the
number of plasma membrane clathrin-coated pits and the concentration of
their structural components were invariant when comparing cells
expressing saturating levels of the chimeric receptors to nonexpressing
cells or to cells expressing only the Tac reporter lacking cytoplasmic
internalization signals. Biochemical analysis showed that the
distribution of coat proteins between assembled coated pits and soluble
pools was also not altered by receptor overexpression. Finally, the cellular localizations of AP2 and AP1 were similarly unaffected. These
results provide a clear indication that receptor endocytic signals do
not determine coated pit levels 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 docking sites that act independently of
endocytic signals.
Department of Pathology and Laboratory Medicine,
University of Pennsylvania School of Medicine, Philadelphia,
Pennsylvania 19104
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