Supplemental Online Material. The mouse embryonic fibroblast cell line #10 derived from β-arrestin1 -/- -β-arrestin2 -/-

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1 # s 1 Supplemental Online Material Materials and Methods Cell lines and tissue culture The mouse embryonic fibroblast cell line #10 derived from β-arrestin1 -/- -β-arrestin2 -/- knock-out animals (S1) was stably transfected with either pcdna3.1-zeo (Invitrogen) or a pcdna3.1-zeo-βarrestin1-flag construct using LipofectAmine (GIBCO-BRL) following the manufacturer s instructions, and maintained in Dulbecco s modified Eagle medium containing 10% fetal bovine serum with 1x penicillin/streptomycin, under selection with 300µg/ml zeocin. Maintenance of COS-7 and Rat-1 cells, and HEK293 cell lines stably overexpressing either FLAG-β 2 -adrenergic receptor or FLAG-β 2 - adrenergic receptor-gfp, are all described previously (S2,S3). Plasmids Mammalian expression constructs of rat and human cdnas for PDE4A4, PDE4B1, PDE4B2, PDE4C2, PDE4D isoforms 1 to 5, rat βarrestin1-flag, rat βarrestin2-flag, are described previously (S4,S5). The catalytically inactive human PDE4D5(D556A) mutant was made using QuickChange (Stratagene) and was confirmed by dideoxynucleotide sequencing. Bacterial expression constructs of β-arrestin1(his) 6, MBP-PDE4D3, MBP-PDE4D5 and MBP are described previously (S6,S7). Yeast 2-

2 # s 2 hybrid constructs of β-arrestin1, β-arrestin2, PDE4D3NT, PDE4D3CT are described previously (S5,S8); yeast transformations and maintenance were performed following the manufacturer s instructions (Clontech). Transfections, co-immunoprecipitations and preparation of membranes Transfection of COS-7 cells was performed using LipofectAmine (GIBCO-BRL), and HEK293 cells using Fugene 6 (Roche) or Polyfect (Qiagen) following the manufacturer s instructions. Cells were harvested in lysis buffer (S5) 48 hours after transfection, cell debris was removed by centrifugation and protein concentrations were normalized. Immunoprecipitations were performed on 2 mg cellular protein for 2 hours at 4 C with 20 µl anti-flag M2 antibody conjugated to agarose beads (Sigma-Aldrich). Samples were washed 3 times with lysis buffer before the proteins were solubilized in Laemmli buffer. Co-immunoprecipitation of endogenous proteins was carried out in Rat-1 cells for clarity, as these cells only express a single PDE4D isoform, PDE4D3 (S9). Immunoprecipitation was carried out from 200 µg cytosolic proteins prepared in KHEM buffer (S10), using 5 µl anti-β-arrestin1 A1CT rabbit polyclonal antiserum or preimmune serum, 50 µl protein A and protein G beads (GIBCO-BRL and Amersham-Pharmacia, respectively) for 2 hours at 4 C. Samples were washed 3 times with KHEM and solubilized in Laemmli buffer. Proteins were separated by PAGE and transferred on to nitrocellulose; western blotting was performed as described previously (S5). Membrane preparations were made as described previously (S10).

3 # s 3 MBP fusion protein pull-down assays Expression and purification of MBP-PDE4D3, MBP-PDE4D5 and MBP are described previously (S7), however, the proteins were not eluted from amylose resin prior to use. Pull-down assays were performed with 500ng of each fusion protein or MBP alone attached to amylose resin (New England Biolabs), incubated with nM βarrestin1(his) 6 in binding buffer (S11) for 2 hours at 4 C. Samples were washed 5 times before solubilizing with Laemmli buffer and processed as described for immunoprecipitations. Phosphodiesterase and protein kinase assays Both assays were performed using the previously described methods (S12,S13), using protein concentrations that gave results within the linear limits of the assay conditions. PDE4 activity was measured as the camp-specific phosphodiesterase activity inhibited by 10µM rolipram. The maximal protein kinase A activity in a sample was determined by the level of Kemptide phosphorylation achieved after the addition of 10µM camp.

4 # s 4 Supplemental Figures (S1), β-arrestin1 binding to PDE4 variants. Lysates from COS7 cells expressing PDEs 4A4, 4B1, 4B2 or 4C2, alone or with β-arrestin1-flag, were subjected to immunoprecipitation with anti-flag M2 affinity agarose. β-arrestin1 and associated PDEs were detected in the immune complexes (upper 2 panels) or cell lysates (lower 2 panels) by immunoblotting with the indicated antibodies. Shown are representative blots of at least 2 experiments.

5 # s 5 (S2), Effect of PDE4D5(D556A) expression on cytosolic PKA activity. Cells were treated in an identical manner to those in Fig. 4C, before stimulation (NS) and after stimulation with 10nM (-)isoproterenol (Iso) for 5 minutes. PKA activity is expressed as % of total PKA activity stimulated with 10µM camp (mean ±S.E.M. of 4 experiments).

6 # s 6 Supplemental Tables Table S1. Interaction of β-arrestin1 and β-arrestin2 with PDE4D3 fragments. Residues and of PDE4D3 were tested for binding to the β-arrestins in the yeast strain PJ69-4A by 2-hybrid analysis. Co-transformation of both expression vectors was confirmed by plating on Trp/Leu-free medium plates and interactions between the proteins were visualized by growth on adenine/trp/leu-free selective medium plates. Controls included co-transformation with empty vectors and with a bait vector expressing lamin C. pas2.1 construct pgad10 construct Growth on selective media? β-arrestin1 β-arrestin2 empty vector Lamin C empty vector empty vector YES YES

7 # s 7 References and Notes S1. T. A. Kohout, F. S. Lin, S. J. Perry, D. A. Conner, R. J. Lefkowitz, Proc Natl Acad Sci U S A 98, (2001). S2. G. J. Della Rocca, S. Maudsley, Y. Daaka, R. J. Lefkowitz, L. M. Luttrell, J Biol Chem 274, (1999). S3. A. J. McLean, G. Milligan, Br J Pharmacol 130, (2000). S4. G. S. Baillie, S. J. MacKenzie, I. McPhee, M. D. Houslay, Br J Pharmacol 131, (2000). S5. P. H. McDonald et al., Science 290, (2000). S6. H. Attramadal et al., J Biol Chem 267, (1992). S7. S. J. Yarwood, M. R. Steele, G. Scotland, M. D. Houslay, G. B. Bolger, J Biol Chem 274, (1999). S8. J. Lim, G. Pahlke, M. Conti, J Biol Chem 274, (1999). S9. M. D. Houslay, unpublished data. S10. G. B. Bolger et al., Biochem J 328, (1997). S11. S. A. Laporte, R. H. Oakley, J. A. Holt, L. S. Barak, M. G. Caron, J Biol Chem 275, (2000). S12. I. McPhee et al., J Biol Chem 274, (1999). S13. J. D. Corbin, Methods Enzymol 99, (1983).

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