Submitted September 23, 2008
Accepted February 17, 2009
Ligand-induced EpoR internalization is mediated by JAK2 and p85 and is impaired by mutations responsible for primary familial and congenital polycythemia
Rita Sulahian, Ondine Cleaver, and Lily Jun-shen Huang*
Department of Cell Biology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX, United States
Department of Molecular Biology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX, United States
* Corresponding author; email: lily.huang{at}utsouthwestern.edu.
Erythropoietin (Epo) is essential for red blood cell production. Several lines of evidence support a causal role of truncating Epo receptor (EpoR) mutations in primary familial and congenital polycythemia (PFCP). Epo-induced endocytosis of EpoR plays important roles in the down-regulation of EpoR signaling and is the primary means that regulates circulating Epo concentrations. Here we show that cell-surface EpoR is internalized via clathrin-mediated endocytosis. Both JAK2 kinase activity and EpoR cytoplasmic tyrosines are important for ligand-dependent EpoR internalization. Phosphorylated Y429, Y431, and Y479 in the EpoR cytoplasmic domain bind p85 subunit of PI3 kinase upon Epo stimulation and individually is sufficient to mediate Epo-dependent EpoR internalization. Knockdown of p85
and p85
or expression of their dominant-negative forms but not inhibition of PI3 kinase activity dramatically impaired EpoR internalization, indicating that p85 and p85 may recruit proteins in the endocytic machinery upon Epo stimulation. Furthermore, mutated EpoRs from PFCP patients lacking the three important tyrosines do not bind p85 or internalize upon stimulation. Addition of residues encompassing Y429 and Y431 to these truncated receptors restored p85 binding and Epo sensitivity. Our results identify a novel PI3 kinase activity-independent function of p85 in EpoR internalization and support a model that defects of internalization in truncated EpoRs from PFCP patients contribute to Epo hypersensitivity and prolonged signaling.
