Structural Requirements for the Ectodomain Cleavage of Human Cell Surface Macrophage Colony-stimulating Factor

Ping Deng,Carl W Rettenmier,Paul K Pattengale

doi:10.1074/jbc.271.27.16338

Ping Deng, Carl W Rettenmier + Show 1 more

Open Access

https://doi.org/10.1074/jbc.271.27.16338

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Abstract

One form of human macrophage colony-stimulating factor (CSF-1(256), M-CSFalpha) is a member of a restricted set of cell surface transmembrane proteins, which is selected to undergo proteolytic ectodomain cleavage. To determine the substrate requirements for this cleavage, we have constructed a series of mutations in the cytoplasmic tail, transmembrane domain, and juxtamembrane region of CSF-1(256) and stably expressed the mutated genes in NIH 3T3 cells. Our results demonstrate that membrane association of the CSF-1 precursor is required for cleavage of its growth factor ectodomain and furthermore that the juxtamembrane region Pro161-Gln162-Leu163-Gln164-Glu165 (PQLQE) (residues 161-165 of the ectodomain) is an essential determinant of cell surface CSF-1(256) cleavage and that the cleavage site is partially sequence-specific. Furthermore, a mechanism of steric hindrance, which likely involves interference with protease accessibility, is postulated to explain the observed decreases in the cleavage efficiency in certain CSF-1 mutants. Finally, our results strongly suggest that the CSF-1 ectodomain is cleaved at or very near the cell surface by a membrane-associated proteolytic system.

Highlights

Certain cell surface proteins are proteolytically cleaved to release their ectodomains into the extracellular compartment
Ectodomain Cleavage of CSF-1256 Requires Membrane Association—The CSF-1256 precursor consists of an amino-terminal signal peptide, a growth factor domain, a small juxtamembrane region, and a transmembrane domain (TM) followed by a short cytoplasmic tail (Fig. 1A)
Similar to the cleavage of wild type (WT) CSF-1256, it was cleaved slowly under basal conditions, and the cleavage was accelerated by the addition of phorbol 12-myristate 13-acetate (PMA) (Fig. 1B)

Summary

Introduction

Certain cell surface proteins are proteolytically cleaved to release their ectodomains into the extracellular compartment. We show that cell membrane association of CSF-1256 is required for its cleavage and that the CSF-1 juxtamembrane region (residues 150 –165) is essential in determining the cleavage.

Results

Conclusion