A growth hormone receptor SNP promotes lung cancer by impairment of SOCS2-mediated degradation

Both humans and mice lacking functional growth hormone (GH) receptors are known to be resistant to cancer. Further, autocrine GH has been reported to act as a cancer promoter. Here we present the first example of a variant of the GH receptor (GHR) associated with cancer promotion, in this case lung cancer. We show that the GHRP495T variant located in the receptor intracellular domain is able to prolong the GH signal in vitro using stably expressing mouse pro-B-cell and human lung cell lines. This is relevant because GH secretion is pulsatile, and extending the signal duration makes it resemble autocrine GH action. Signal duration for the activated GHR is primarily controlled by suppressor of cytokine signalling 2 (SOCS2), the substrate recognition component of the E3 protein ligase responsible for ubiquitinylation and degradation of the GHR. SOCS2 is induced by a GH pulse and we show that SOCS2 binding to the GHR is impaired by a threonine substitution at Pro 495. This results in decreased internalisation and degradation of the receptor evident in TIRF microscopy and by measurement of mature (surface) receptor expression. Mutational analysis showed that the residue at position 495 impairs SOCS2 binding only when a threonine is present, consistent with interference with the adjacent Thr494. The latter is key for SOCS2 binding, together with nearby Tyr487, which must be phosphorylated for SOCS2 binding. We also undertook nuclear magnetic resonance spectroscopy approach for structural comparison of the SOCS2 binding scaffold Ile455-Ser588, and concluded that this single substitution has altered the structure of the SOCS2 binding site. Importantly, we find that lung BEAS-2B cells expressing GHRP495T display increased expression of transcripts associated with tumour proliferation, epithelial–mesenchymal transition and metastases (TWIST1, SNAI2, EGFR, MYC and CCND1) at 2 h after a GH pulse. This is consistent with prolonged GH signalling acting to promote cancer progression in lung cancer

Order and disorder - An integrative structure of the full-length human growth hormone receptor

Because of its small size (70 kilodalton) and large content of structural disorder (>50%), the human growth hormone receptor (hGHR) falls between the cracks of conventional high-resolution structural biology methods. Here, we study the structure of the full-length hGHR in nanodiscs with small-angle x-ray scattering (SAXS) as the foundation. We develop an approach that combines SAXS, x-ray diffraction, and NMR spectroscopy data obtained on individual domains and integrate these through molecular dynamics simulations to interpret SAXS data on the full-length hGHR in nanodiscs. The hGHR domains reorient freely, resulting in a broad structural ensemble, emphasizing the need to take an ensemble view on signaling of relevance to disease states. The structure provides the first experimental model of any full-length cytokine receptor in a lipid membrane and exemplifies how integrating experimental data from several techniques computationally may access structures of membrane proteins with long, disordered regions, a widespread phenomenon in biology

Light-Induced Rearrangement of the β5 Strand in the BLUF Photoreceptor SyPixD (Slr1694)

The structural changes that facilitate signal transduction in blue light sensors using FAD (BLUF) photoreceptors and confer the stability of the rearranged hydrogen bond network between flavin and protein in the signaling state are still poorly understood. Here, we investigate a semiconserved Trp residue in SyPixD (Slr1694) by isotope-edited vibrational spectroscopy and site-directed mutagenesis. In the signaling state, a β-sheet structure involving the backbone of W91 is formed without apparent change of environment of the W91 indole side chain. Mutation of W91, however, significantly influences the stability of the light-adapted state, suggesting that backbone rigidity rather than discrete side-chain conformations govern the stability of the light-adapted state. On the basis of computational and crystallographic models, we interpret these changes as a +1 register shift of the β2/β5 interaction with an unaffected indole side-chain conformation, rather than a +2 register shift accompanied by an indole side-chain flip that was previously proposed on the basis of X-ray structures