The International Journal of Biochemistry & Cell Biology 44 (2012) 290–301 Contents lists available at SciVerse ScienceDirect The International Journal of Biochemistry & Cell Biology jo ur nal homep ag e: www.elsevier.com/locate/biocel Comparative proteomic analysis to dissect differences in signal transduction in activating TSH receptor mutations in the thyroid Kerstin Krause a,1 , Alexandra Boisnard a,1 , Christian Ihling b , Marian Ludgate c , Markus Eszlinger a , Knut Krohn a , Andrea Sinz b , Dagmar Fuhrer d,∗ a Clinic for Endocrinology and Nephrology, University of Leipzig, Liebigstr. 20, D-04413 Leipzig, Germany b Department of Pharmaceutical Chemistry & Bioanalytics, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4, D-06120 Halle (Saale), Germany c Centre for Endocrine and Diabetes Sciences, School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK d Department of Endocrinology and Metabolism, Division of Laboratory Research, University Hospital Essen, Hufelandstr. 55, 45147 Essen, Germany a r t i c l e i n f o Article history: Received 17 May 2011 Received in revised form 9 October 2011 Accepted 27 October 2011 Available online 3 November 2011 Keywords: Thyroid hormone stimulating receptor (TSHR) cAMP signaling Toxic thyroid nodules 2D-DIGE proteomics Mass spectrometry a b s t r a c t In the thyroid, cAMP controls both thyroid growth and function. Gain-of-function mutations in the thyroid-stimulating hormone receptor (TSHR) lead to constitutive cAMP formation and are a major cause of autonomous thyroid adenomas. The impact of activating TSHR mutations on the signal transduction network of the thyrocyte is not fully understood. To gain more insights into constitutive TSHR signaling, rat thyrocytes (FRTL-5 cells) with stable expres- sion of three activating TSHR mutants (mutTSHR: A623I, L629F and Del613-621), which differ in their functional characteristics in vitro, were analyzed by a quantitative proteomic approach and compared to the wild-type TSHR (WT-TSHR). This study revealed (1) differences in the expression of Rab proteins suggesting an increased TSHR internalization in mutTSHR but not in the WT-TSHR; (2) differential stim- ulation of PI3K/Akt signaling in mutTSHR vs. WT-TSHR cells, (3) activation of Epac, impairing short-time Akt phosphorylation in both, mutTSHR and WT-TSHR cells. Based on the analysis of global changes in protein expression patterns, our findings underline the complexity of gain-of-function TSHR signaling in thyrocytes, which extends beyond pure cAMP and/or IP formation. Moreover, evidence for augmented endocytosis in the mutTSHR, adds to a new concept of TSHR signaling in thyroid autonomy. Further studies are required to clarify whether the observed differences in Rab, PI3K and Epac signaling may contribute to differences in the phenotypic presentation, i.e. stimulation of function and growth of thyroid autonomy in vivo. © 2011 Elsevier Ltd. All rights reserved. 1. Introduction The TSH receptor (TSHR) couples to multiple G proteins (Laugwitz et al., 1996) but most of the effects of TSH are medi- ated through Gs and cAMP (Kimura et al., 2001; Medina and Santisteban, 2000). cAMP activates multiple downstream targets, including PKA and guanine nucleotide exchange factors for Rap (de Rooij et al., 1998; Kawasaki et al., 1998a) and Ras (Pak et al., 2002; Pham et al., 2000). Besides the control of differentiated functions, cAMP inhibits or stimulates cell proliferation depending on the cell Abbreviations: PKA, protein kinase A; GEF(s), guanine nucleotide exchange factor(s); EPAC, exchange proteins activated by cAMP; MAPK, mitogen activated pro- tein kinase; mTOR, mammalian target of rapamycin; 8-CPT, 8-CPT-2-O-Me-cAMP. ∗ Corresponding author. Tel.: +49 201 723 2821. E-mail address: Dagmar.fuehrer@uk-essen.de (D. Fuhrer). 1 These authors contributed equally to this study. type. In the thyroid, chronic cAMP stimulation leads to toxic thyroid hyperplasia in transgenic mice (e.g. due to adenosine A2 receptor overexpression) as well as hyperthyroidism and goiter in individ- uals with germline gain-of-function TSHR mutations (Vassart and Dumont, 1992; Paschke and Ludgate, 1997; Corvilain et al., 2001). In addition, presence of TSHR autoantibodies also results in the chronic activation of the TSHR (Morshed et al., 2009). Somatic acti- vating TSHR mutations can be found in 60% of patients with toxic thyroid nodules. Functional characterization of TSHR mutants is usually performed in COS-7 cells, a non-thyroid cell system, and shows that the mutants differ in their potency to activate G and Gq signaling. The in vitro findings however do not correlate with the biological behaviour of the respective mutants in vivo. We have pre- viously shown that the biological effects of TSHR mutations must be investigated in the correct cellular context and that biological net effects in terms of stimulation of thyroid growth and function cannot be deduced purely on measurement of second messengers (Fuhrer et al., 2003). Thus it is likely that still unknown signaling 1357-2725/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2011.10.024