SOCS2 negatively regulates growth hormone action in vitro and in vivo
Christopher J. Greenhalgh, Elizabeth Rico-Bautista, Mattias Lorentzon, Anne L. Thaus, Phillip O. Morgan, Tracy A. Willson, Panagiota Zervoudakis, Donald Metcalf, Ian Street, Nicos A. Nicola, Andrew D. Nash, Louis J. Fabri, Gunnar Norstedt, Claes Ohlsson, Amilcar Flores-Morales, Warren S. Alexander, Douglas J. Hilton
Christopher J. Greenhalgh, Elizabeth Rico-Bautista, Mattias Lorentzon, Anne L. Thaus, Phillip O. Morgan, Tracy A. Willson, Panagiota Zervoudakis, Donald Metcalf, Ian Street, Nicos A. Nicola, Andrew D. Nash, Louis J. Fabri, Gunnar Norstedt, Claes Ohlsson, Amilcar Flores-Morales, Warren S. Alexander, Douglas J. Hilton
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Article Endocrinology

SOCS2 negatively regulates growth hormone action in vitro and in vivo

Abstract

Mice deficient in SOCS2 display an excessive growth phenotype characterized by a 30–50% increase in mature body size. Here we show that the SOCS2–/– phenotype is dependent upon the presence of endogenous growth hormone (GH) and that treatment with exogenous GH induced excessive growth in mice lacking both endogenous GH and SOCS2. This was reflected in terms of overall body weight, body and bone lengths, and the weight of internal organs and tissues. A heightened response to GH was also measured by examining GH-responsive genes expressed in the liver after exogenous GH administration. To further understand the link between SOCS2 and the GH-signaling cascade, we investigated the nature of these interactions using structure/function and biochemical interaction studies. Analysis of the 3 structural motifs of the SOCS2 molecule revealed that each plays a crucial role in SOCS2 function, with the conserved SOCS-box motif being essential for all inhibitory function. SOCS2 was found to bind 2 phosphorylated tyrosines on the GH receptor, and mutational analysis of these amino acids showed that both were essential for SOCS2 function. Together, the data provide clear evidence that SOCS2 is a negative regulator of GH signaling.

Authors

Christopher J. Greenhalgh, Elizabeth Rico-Bautista, Mattias Lorentzon, Anne L. Thaus, Phillip O. Morgan, Tracy A. Willson, Panagiota Zervoudakis, Donald Metcalf, Ian Street, Nicos A. Nicola, Andrew D. Nash, Louis J. Fabri, Gunnar Norstedt, Claes Ohlsson, Amilcar Flores-Morales, Warren S. Alexander, Douglas J. Hilton

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SOCS2 motif control of GH signaling. (A) A schematic diagram of SOCS2 is...
SOCS2 motif control of GH signaling. (A) A schematic diagram of SOCS2 is provided to help clarify mutant constructs used and residues mutated in the SH2 domain. (B–D) 293T cells were transfected with pig GH receptor and SOCS2 (SOCS2 WT), or with the following SOCS2 mutant constructs: (B) SOCS2 with a point mutation in the SH2 domain R73K (SOCS2 D) or SOCS2 with a triple mutation at R73K, D74E, and S75C in the SH2 domain (SOCS2 KD); (C) SOCS2 lacking the 37-AA N terminus (SOCS2°NT) or SOCS2 with the N-terminal region of SOCS1 (SOCS1/2/2); or (D) SOCS2 lacking the 39-AA C terminus (SOCS2°SB) at a range of plasmid concentrations (ng). The transfected cells were then stimulated with rpGH and the luciferase activity from an LHRE-luciferase reporter was measured. Data is corrected for transfection efficiency by cotransfection of a β-galactosidase–expressing plasmid. Luciferase activity was corrected using values obtained in the absence of GH, then expressed as a percentage of wild-type activity, which was assigned a value of 100%. Experiments were performed in triplicate, and data presented here are representative of 3 independent experiments. (E) Flag-tagged SOCS2 and empty vector were transfected into 293T cells, lysed, and immunoprecipitated using antibodies against Flag. After separation on SDS-PAGE and Western transfer, blots were probed with antibodies against Elongins B and C, then stripped and reprobed with antibodies against the Flag epitope.