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Molecular and Cellular Biology, September 2008, p. 5275-5287, Vol. 28, No. 17
0270-7306/08/$08.00+0     doi:10.1128/MCB.00350-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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

Bentley Varghese,^1,2, Herve Barriere,^3, Christopher J. Carbone,^1, Anamika Banerjee,¹ Gayathri Swaminathan,¹ Alexander Plotnikov,¹ Ping Xu,⁴ Junmin Peng,⁴ Vincent Goffin,⁵ Gergely L. Lukacs,³ and Serge Y. Fuchs^1*

Department of Animal Biology and Mari Lowe Comparative Oncology Center, University of Pennsylvania, Philadelphia, Pennsylvania,¹ Cell and Molecular Biology Group, Biomedical Graduate School, University of Pennsylvania, Philadelphia, Pennsylvania,² Department Of Physiology, McGill University, Montreal, Canada,³ Department of Human Genetics, Center for Neurodegenerative Disease, Emory University, Atlanta, Georgia,⁴ INSERM, U845, Centre de Recherche Croissance et Signalisation, Equipe PRL, GH and Tumours, and Université Paris Descartes, Faculté de Médecine, Paris F-75015, France⁵

Received 29 February 2008/ Returned for modification 28 March 2008/ Accepted 16 June 2008

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.

Published ahead of print on 23 June 2008.

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

B.V., H.B., and C.J.C. contributed equally to the manuscript.