Polyubiquitination of Prolactin Receptor Stimulates Its Internalization, Postinternalization Sorting, and Degradation via the Lysosomal Pathway

Abstract

The ubiquitination of the receptor that mediates signaling induced by the polypeptide pituitary hormone prolactin (PRL) has been shown to lead to the degradation of this receptor and to the ensuing negative regulation of cellular responses to PRL. However, the mechanisms of PRL receptor (PRLr) proteolysis remain largely to be determined. Here we provide evidence that PRLr is internalized and primarily degraded via the lysosomal pathway. Ubiquitination of PRLr is essential for the rapid internalization of PRLr, which proceeds through a pathway dependent on clathrin and the assembly polypeptide 2 (AP2) adaptor complexes. Recruitment of AP2 to PRLr is stimulated by PRLr ubiquitination, which also is required for the targeting of already internalized PRLr to the lysosomal compartment. While mass spectrometry analysis revealed that both monoubiquitination and polyubiquitination (via both K48- and K63-linked chains) occur on PRLr, the results of experiments using forced expression of ubiquitin mutants indicate that PRLr polyubiquitination via K63-linked chains is important for efficient interaction of PRLr with AP2 as well as for efficient internalization, postinternalization sorting, and proteolytic turnover of PRLr. We discuss how specific ubiquitination may regulate early and late stages of endocytosis of PRLr and of related receptors to contribute to the negative regulation of the magnitude and duration of downstream signaling.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://mcb.asm.org .

ACKNOWLEDGMENTS

We thank A. Eastman, Y. Yarden, Z. Ronai, and A. F. Parlow for reagents, L. Schuler for discussion of the results, and I. Fernandez for critical comments on the manuscript.

This work was supported by NIH grants CA115281 (to S.Y.F.) and AG025688 (to J.P.), by the Canadian Cystic Fibrosis Foundation (H.B.), by grants from CIHR and NIDDK (to G.L.L.), and by funding from Institut National de la Santé et de la Recherche Médicale (to V.G.).