Tryptophan at the transmembrane-cytosolic junction modulates thrombopoietin receptor dimerization and activation.

Dimerization of single-pass membrane receptors is essential for activation. In the human thrombopoietin receptor (TpoR), a unique amphipathic RWQFP motif separates the transmembrane (TM) and intracellular domains. Using a combination of mutagenesis, spectroscopy, and biochemical assays, we show that W515 of this motif impairs dimerization of the upstream TpoR TM helix. TpoR is unusual in that a specific residue is required for this inhibitory function, which prevents receptor self-activation. Mutations as diverse as W515K and W515L cause oncogenic activation of TpoR and lead to human myeloproliferative neoplasms. Two lines of evidence support a general mechanism in which W515 at the intracellular juxtamembrane boundary inhibits dimerization of the TpoR TM helix by increasing the helix tilt angle relative to the membrane bilayer normal, which prevents the formation of stabilizing TM dimer contacts. First, measurements using polarized infrared spectroscopy show that the isolated TM domain of the active W515K mutant has a helix tilt angle closer to the bilayer normal than that of the wild-type receptor. Second, we identify second-site R514W and Q516W mutations that reverse dimerization and tilt angle changes induced by the W515K and W515L mutations. The second-site mutations prevent constitutive activation of TpoR W515K/L, while preserving ligand-induced signaling. The ability of tryptophan to influence the angle and dimerization of the TM helix in wild-type TpoR and in the second-site revertants is likely associated with its strong preference to be buried in the headgroup region of membrane bilayers.

Synthesis, purification, and characterization of single helix membrane peptides and proteins for NMR spectroscopy.

Membrane proteins function as receptors, channels, transporters, and enzymes. These proteins are generally difficult to express and purify in a functional form due to the hydrophobic nature of their membrane spanning sequences. Studies on membrane proteins with a single membrane spanning helix have been particularly challenging. Single-pass membrane proteins will often form dimers or higher order oligomers in cell membranes as a result of sequence motifs that mediate specific transmembrane helix interactions. Understanding the structural basis for helix association provides insights into how these proteins function. Nevertheless, nonspecific association or aggregation of hydrophobic membrane spanning sequences can occur when isolated transmembrane domains are reconstituted into membrane bilayers or solubilized into detergent micelles for structural studies by solid-state or solution NMR spectroscopy. Here, we outline the methods used to synthesize, purify, and characterize single transmembrane segments for structural studies. Two synthetic strategies are discussed. The first strategy is to express hydrophobic peptides as protein chimera attached to the maltose binding protein. The second strategy is by direct chemical synthesis. Purification is carried out by several complementary chromatography methods. The peptides are solubilized in detergent for solution NMR studies or reconstituted into model membranes for solid-state NMR studies. We describe the methods used to characterize the reconstitution of these systems prior to NMR structural studies to establish if there is nonspecific aggregation.

Orientation-specific signalling by thrombopoietin receptor dimers.

Ligand binding to the thrombopoietin receptor is thought to stabilize an active receptor dimer that regulates megakaryocyte differentiation and platelet formation, as well as haematopoietic stem cell renewal. By fusing a dimeric coiled coil in all seven possible orientations to the thrombopoietin receptor transmembrane (TM)-cytoplasmic domains, we show that specific biological effects and in vivo phenotypes are imparted by distinct dimeric orientations, which can be visualized by cysteine mutagenesis and crosslinking. Using functional assays and computational searches, we identify one orientation that represents the inactive dimeric state and another similar to a physiologically activated receptor. Several other dimeric orientations are identified that induce proliferation and in vivo myeloproliferative and myelodysplastic disorders, indicating the receptor can signal from several dimeric interfaces. The set of dimeric thrombopoietin receptors with different TM orientations may offer new insights into the activation of distinct signalling pathways by a single receptor and suggests that subtle differences in cytokine receptor dimerization provide a new layer of signalling regulation that is relevant for disease.

An activating mutation in the CSF3R gene induces a hereditary chronic neutrophilia.

We identify an autosomal mutation in the CSF3R gene in a family with a chronic neutrophilia. This T617N mutation energetically favors dimerization of the granulocyte colony-stimulating factor (G-CSF) receptor transmembrane domain, and thus, strongly promotes constitutive activation of the receptor and hypersensitivity to G-CSF for proliferation and differentiation, which ultimately leads to chronic neutrophilia. Mutant hematopoietic stem cells yield a myeloproliferative-like disorder in xenotransplantation and syngenic mouse bone marrow engraftment assays. The survey of 12 affected individuals during three generations indicates that only one patient had a myelodysplastic syndrome. Our data thus indicate that mutations in the CSF3R gene can be responsible for hereditary neutrophilia mimicking a myeloproliferative disorder.