Transforming growth factor-beta receptors interact with AP2 by direct binding to beta2 subunit.
Transforming growth factor-beta (TGF-beta) superfamily members regulate a wide range of biological processes by binding to two transmembrane serine/threonine kinase receptors, type I and type II. We have previously shown that the internalization of these receptors is inhibited by K(+) depletion, cytosol acidification, or hypertonic medium, suggesting the involvement of clathrin-coated pits. However, the involvement of the clathrin-associated adaptor complex AP2 and the identity of the AP2 subunit that binds the receptors were not known. Herein, we have studied these issues by combining studies on intact cells with in vitro assays. Using fluorescence photobleaching recovery to measure the lateral mobility of the receptors on live cells (untreated or treated to alter their coated pit structure), we demonstrated that their mobility is restricted by interactions with coated pits. These interactions were transient and mediated through the receptors' cytoplasmic tails. To measure direct binding of the receptors to specific AP2 subunits, we used yeast two-hybrid screens and in vitro biochemical assays. In contrast to most other plasma membrane receptors that bind to AP2 via the mu2 subunit, AP2/TGF-beta receptor binding was mediated by a direct interaction between the beta2-adaptin N-terminal trunk domain and the cytoplasmic tails of the receptors; no binding was observed to the mu2, alpha, or sigma2 subunits of AP2 or to mu1 of AP1. The data uniquely demonstrate both in vivo and in vitro the ability of beta2-adaptin to directly couple TGF-beta receptors to AP2 and to clathrin-coated pits, providing the first in vivo evidence for interactions of a transmembrane receptor with beta2-adaptin.
Yao
Diying
D
Department of Biochemistry and Molecular Biology and Thoracic Diseases Research Unit, Mayo Clinic, Rochester, Minnesota 55905, USA.
Ehrlich
Marcelo
M
Henis
Yoav I
YI
Leof
Edward B
EB
D033962:Adaptor Protein Complex 2 / Q000235:genetics / Q000378:metabolism*; D033966:Adaptor Protein Complex beta Subunits / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D019556:COS Cells; D002746:Chlorpromazine / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology; D036681:Fluorescence Recovery After Photobleaching; D011485:Protein Binding; D021122:Protein Subunits / Q000378:metabolism*; D018125:Receptors, Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D033962:Adaptor Protein Complex 2; D033966:Adaptor Protein Complex beta Subunits; D000906:Antibodies; D002966:Clathrin; D021122:Protein Subunits; D018125:Receptors, Transforming Growth Factor beta; D011993:Recombinant Fusion Proteins; D002746:Chlorpromazine