Systematic Mutational Analysis of the Cation-independent Mannose 6-Phosphate/Insulin-like Growth Factor II Receptor Cytoplasmic Domain

doi:10.1074/jbc.272.11.7003

Systematic Mutational Analysis of the Cation-independent Mannose 6-Phosphate/Insulin-like Growth Factor II Receptor Cytoplasmic Domain

AN ACIDIC CLUSTER CONTAINING A KEY ASPARTATE IS IMPORTANT FOR FUNCTION IN LYSOSOMAL ENZYME SORTING*

From the ^‡ Center for Advanced Biotechnology and Medicine, the
^§ Graduate Program in Microbiology and Molecular Genetics, Rutgers University, and the
^∥ Department of Pharmacology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854

** To whom correspondence should be addressed. Tel.: 908-235-5360; Fax: 908-235-4850.

↵^¶ Present address: Div. of Biology, California Institute of Technology, Pasadena, CA 91125.

Abstract

We have used systematic mutational analysis to identify signals in the 166-residue murine cation-independent mannose 6-phosphate/insulin-like growth factor II receptor cytoplasmic domain required for efficient sorting of lysosomal enzymes. Alanine cluster mutagenesis on all conserved residues apart from the endocytosis signal demonstrates that the major sorting determinant is a conserved casein kinase II site followed by a dileucine motif (¹⁵⁷DDSDEDLL¹⁶⁴). Small deletions or additions outside this region have severe to mild effects, indicating that context is important. Single residue mutagenesis indicates that cycles of serine phosphorylation/dephosphorylation are not obligatory for sorting. In addition, the two leucine residues and four of the five negatively charged residues can readily tolerate conservative substitutions. In contrast, aspartate 160 could not tolerate isoelectric or isosteric substitutions, implicating it as a critical component of the sorting signal.

Footnotes

↵* This work was supported by National Institutes of Health Grant DK-45992. 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:

M6P/IGF2R

mannose 6-phosphate/insulin-like growth factor II receptor

FBS

fetal bovine serum

DMEM

Dulbecco's modified Eagle's medium

PBS

phosphate-buffered saline.
- Received August 13, 1996.
- Revision received December 27, 1996.