Active and Inactive Orientations of the Transmembrane and Cytosolic Domains of the Erythropoietin Receptor Dimer

Abstract

Binding of erythropoietin to the erythropoietin receptor (EpoR) extracellular domain orients the transmembrane (TM) and cytosolic regions of the receptor dimer into an unknown activated conformation. By replacing the EpoR extracellular domain with a dimeric coiled coil, we engineered TM EpoR fusion proteins where the helical TM domains were constrained into seven possible relative orientations. We identify one dimeric TM conformation that imparts full activity to the cytosolic domain of the receptor and signals via JAK2, STAT proteins, and MAP kinase, one partially active orientation that preferentially activates MAP kinase, and one conformation corresponding to the inactive receptor. The active and inactive conformations were independently identified by computational searches for low-energy TM dimeric structures. We propose a specific EpoR-activated interface and suggest its use for structural and signaling studies.

S179D prolactin: Antagonistic agony!

“…Each study showed a one amino acid twist in the transmembrane helical region of the receptor to produce altered signaling. In the case of the erythropoietin receptor, different degrees of rotation produced altered ratios of Jak2-Stat5 to ERK 1/2 signaling (Seubert et al, 2003), just as we see for unmodified PRL versus S179D PRL. Also, a naturally-occurring variant of growth hormone with a one amino acid substitution has altered signaling at the receptor (Lewis et al, 2004).…”

“…Instead, as previously illustrated (Walker, 2005) and as shown in figure 2, rotation of the receptor may well be the key. The potential for such a scenario is supported by some elegant work using the erythropoietin receptor (Seubert et al, 2003) and, more recently using the growth hormone receptor (Brown et al, 2005). Each study showed a one amino acid twist in the transmembrane helical region of the receptor to produce altered signaling.…”

“…It therefore appears that S179D PRL has a different conformation and that its interaction with a pair of PRLRs may produce a different conformation in them. This altered conformation versus unmodified PRL may well be subtle since based on the work with the erythropoietin receptor, an approximately one amino acid turn in the transmembrane domain would be sufficient to alter signaling from primarily Jak-Stat to primarily ERK (Seubert et al, 2003). As cartooned in figure 2, the long PRLR responds differently to the two forms of PRL and this difference is amplified once the short PRLRs are upregulated by the promotion of alternative splicing.…”

JAK kinases overexpression promotes in vitro cell transformation

“…For retroviral expression, wild-type and mutated mJAK1, mJAK2, hJAK3 and hTYK2 cDNAs were subcloned into the pMX-IRES-CD2 or pMX-IRES-CD4 biscistronic retroviral vector upstream of the IRES (Liu et al, 2000). Retroviral supernatants were generated by transient transfection of the BOSC packaging cell line with biscistronic vectors encoding the gene of interest and CD2 or CD4 as markers, and used for infection, as described (Liu et al, 2000;Seubert et al, 2003). Populations of cells expressing CD2 or CD4 above a predetermined level (top 25%) were isolated by FACS sorting.…”

“…This was illustrated by studies of fusion proteins composed of dimeric-coiled coil and the cytosolic part of the EPO receptor, which allow to identify active and inactive conformation of the receptor dimer. Overexpression of JAK2 allowed the activation of cell signaling with constructs in suboptimal conformation (Seubert et al, 2003). Higher efficiency of signal transduction due to higher concentration of JAK molecules could also increase cytokine responsiveness.…”