Structural Determinants of Interaction of Tyrosine-based Sorting Signals with the Adaptor Medium Chains

doi:10.1074/jbc.271.46.29009

Structural Determinants of Interaction of Tyrosine-based Sorting Signals with the Adaptor Medium Chains*

From the Cell Biology and Metabolism Branch, NICHD and the
^§ Genetics and Biochemistry Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892

^¶To whom correspondence should be addressed:
CBMB-NICHD, NIH, Bldg. 18T, Rm. 101, 18 Library Dr. MSC 5430, Bethesda, MD 20892-5430
. Tel.: 301-496-6368; Fax: 301-402-0078.

Abstract

Many integral membrane proteins contain tyrosine-based signals within their cytoplasmic domains that mediate internalization from the cell surface and targeting to lysosomal compartments. Internalization depends on an interaction of the tyrosine-based signals with the clathrin-associated adaptor complex AP-2 at the plasma membrane, whereas lysosomal targeting involves interaction of the signals with an analogous complex, AP-1, at the trans-Golgi network. Recent studies have identified the medium chains μ₂ of AP-2 and μ₁ of AP-1 as the recognition molecules for tyrosine-based signals. We have now investigated the structural determinants for interaction of the signals with μ₂ and μ₁. The position of the signals was found to be an important determinant of interactions with μ₂ and μ₁; signals were most effective when present at the carboxyl terminus of a polypeptide sequence. Another important determinant of interactions was the identity of residues surrounding the critical tyrosine residue. Mutation of some residues affected interactions with μ₂ and μ₁ similarly, whereas other mutations had differential effects. These observations suggest that both the position and the exact sequence of tyrosine-based sorting signals are major determinants of selectivity in their interaction with clathrin-associated adaptor complexes.

Footnotes

↵^‡ Supported by a fellowship from the Japan Society for the Promotion of Science.
↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

TGN

trans-Golgi network

GAL4bd

GAL4 DNA-binding domain

GAL4ad

GAL4 transcription activation domain

GST

glutathione S-transferase.
↵²Notice that the β-galactosidase activity values in Figs. 2, 3, 4, 5, 6 are represented on a logarithmic scale.
- Received June 25, 1996.
- Revision received September 6, 1996.