Arrestin/clathrin interaction. Localization of the arrestin binding locus to the clathrin terminal domain.
Previously we demonstrated that nonvisual arrestins exhibit a high affinity interaction with clathrin, consistent with an adaptor function in the internalization of G protein-coupled receptors (Goodman, O. B., Jr., Krupnick, J. G., Santini, F., Gurevich, V. V., Penn, R. B., Gagnon, A. W., Keen, J. H., and Benovic, J. L. (1996) Nature 383, 447-450). In this report we show that a short sequence of highly conserved residues within the globular clathrin terminal domain is responsible for arrestin binding. Limited proteolysis of clathrin cages results in the release of terminal domains and concomitant abrogation of arrestin binding. The nonvisual arrestins, beta-arrestin and arrestin3, but not visual arrestin, bind specifically to a glutathione S-transferase-clathrin terminal domain fusion protein. Deletion analysis and alanine scanning mutagenesis localize the binding site to residues 89-100 of the clathrin heavy chain and indicate that residues 1-100 can function as an independent arrestin binding domain. Site-directed mutagenesis identifies an invariant glutamine (Glu-89) and two highly conserved lysines (Lys-96 and Lys-98) as residues critical for arrestin binding, complementing hydrophobic and acidic residues in arrestin3 which have been implicated in clathrin binding (Krupnick, J. G., Goodman, O. B., Jr., Keen, J. H., and Benovic, J. L. (1997) J. Biol. Chem. 272, 15011-15016). Despite exhibiting high affinity clathrin binding, arrestins do not induce coat assembly. The terminal domain is oriented toward the plasma membrane in coated pits, and its binding of both arrestins and AP-2 suggests that this domain is the anchor responsible for adaptor-receptor recruitment to the coated pit.
Goodman
O B
OB
Department of Biochemistry, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Krupnick
J G
JG
Gurevich
V V
VV
Benovic
J L
JL
Keen
J H
JH
D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D019393:Arrestin / Q000737:chemistry* / Q000378:metabolism*; D019390:Arrestins / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D002966:Clathrin / Q000737:chemistry* / Q000378:metabolism*; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004926:Escherichia coli; D005973:Glutamine; D005982:Glutathione Transferase; D008239:Lysine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017384:Sequence Deletion; D000071557:beta-Arrestins
D019393:Arrestin; D019390:Arrestins; D002966:Clathrin; D011993:Recombinant Fusion Proteins; C082333:arrestin3; D000071557:beta-Arrestins; D005973:Glutamine; D005982:Glutathione Transferase; D008239:Lysine; D000409:Alanine