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SUPPLEMENTARY DATA SD-Figure 1 Comparison by western blot analysis of wild type and mutant CRH-R1α receptor expression in HEK293 cell membranes. Cell membrane proteins from HEK293 cells transiently expressing wild type or mutant CRH-R1α receptors were prepared using ProteoExtract® Native Membrane Protein Extraction Kit as described in Experimental Procedures and were fractionated on SDS-PAGE, transferred to and subjected to immunoblotting with a specific CRH-R1/2 antibody and IRDye™ 800 secondary antibody, using the Odyssey detection system, or immunoblotted with a anti-cadherin antibody and visualized with ECL. Representative Western blots are shown: identical results were obtained from 3 independent transfection experiments. Similar results were obtained when mutants R1αI293A, R1αK297A, R1αR299A and R1αR310A were transiently expressed in HEK293 cells (data not shown)

SD-Figure 2 Effect of Gi-protein activators/inhibitors on Ucn1-induced cAMP release from HEK293 cells expressing wild type CRH-R1α receptors. HEK293 cells, transiently expressing wild type CRH-R1α as described in Experimental Procedures, were pre-treated with or without (a) pertussis-toxin (PTX) for 12 h, (final concentration, 100 ng/ml) or mastoparan the wasp venom-derived peptide (20 µM for 1h), to inactivate or activate Gi/o-proteins respectively, before stimulation with different concentrations of Ucn1 (0.1-1000nM) for 15min cells. In some experiments, cells overexpressing a dominant-negative Gαi(Q205L/D273N) were pre-treated with 20 µM mastoparan (b) or the suramin analogue NF023 (100nM for 2h) (c) prior to stimulation with Ucn1 (100nM) (b). Cyclic AMP was measured by ELISA and responses (measured as Ucn1-induced cAMP fold increase above basal) shown represent the mean ±SEM of 3 estimations from three independent experiments. *, P