Differential regulation of adipocyte PDE3B in distinct membrane compartments by insulin and the beta3-adrenergic receptor agonist CL316243: effects of caveolin-1 knockdown on formation/maintenance of macromolecular signalling complexes.
In adipocytes, PDE3B (phosphodiesterase 3B) is an important regulatory effector in signalling pathways controlled by insulin and cAMP-increasing hormones. Stimulation of 3T3-L1 adipocytes with insulin or the beta3-adrenergic receptor agonist CL316243 (termed CL) indicated that insulin preferentially phosphorylated/activated PDE3B associated with internal membranes (endoplasmic reticulum/Golgi), whereas CL preferentially phosphorylated/activated PDE3B associated with caveolae. siRNA (small interfering RNA)-mediated KD (knockdown) of CAV-1 (caveolin-1) in 3T3-L1 adipocytes resulted in down-regulation of expression of membrane-associated PDE3B. Insulin-induced activation of PDE3B was reduced, whereas CL-mediated activation was almost totally abolished. Similar results were obtained in adipocytes from Cav-1-deficient mice. siRNA-mediated KD of CAV-1 in 3T3-L1 adipocytes also resulted in inhibition of CL-stimulated phosphorylation of HSL (hormone-sensitive lipase) and perilipin A, and of lipolysis. Superose 6 gel-filtration chromatography of solubilized membrane proteins from adipocytes stimulated with insulin or CL demonstrated the reversible assembly of distinct macromolecular complexes that contained 32P-phosphorylated PDE3B and signalling molecules thought to be involved in its activation. Insulin- and CL-induced macromolecular complexes were enriched in cholesterol, and contained certain common signalling proteins [14-3-3, PP2A (protein phosphatase 2A) and cav-1]. The complexes present in insulin-stimulated cells contained tyrosine-phosphorylated IRS-1 (insulin receptor substrate 1) and its downstream signalling proteins, whereas CL-activated complexes contained beta3-adrenergic receptor, PKA-RII [PKA (cAMP-dependent protein kinase)-regulatory subunit] and HSL. Insulin- and CL-mediated macromolecular complex formation was significantly inhibited by CAV-1 KD. These results suggest that cav-1 acts as a molecular chaperone or scaffolding molecule in cholesterol-rich lipid rafts that may be necessary for the proper stabilization and activation of PDE3B in response to CL and insulin.
Ahmad
Faiyaz
F
Translational Medicine Branch, NHLBI, NIH, Bethesda, MD 20892, USA. Ahmadf@nhlbi.nih.gov
Lindh
Rebecka
R
Tang
Yan
Y
Ruishalme
Iida
I
Ost
Anita
A
Sahachartsiri
Bobby
B
Strålfors
Peter
P
Degerman
Eva
E
Manganiello
Vincent C
VC
D041721:3T3-L1 Cells; D017667:Adipocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D000318:Adrenergic beta-Agonists / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D021941:Caveolae / Q000187:drug effects / Q000378:metabolism; D051242:Caveolin 1 / Q000235:genetics / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3 / Q000235:genetics / Q000378:metabolism*; D004149:Dioxoles / Q000494:pharmacology*; D004721:Endoplasmic Reticulum / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic; D006056:Golgi Apparatus / Q000201:enzymology; D007328:Insulin / Q000494:pharmacology*; D008066:Lipolysis / Q000187:drug effects; D046911:Macromolecular Substances / Q000378:metabolism; D051379:Mice; D010766:Phosphorylation / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D013379:Substrate Specificity
D000318:Adrenergic beta-Agonists; D051242:Caveolin 1; D004149:Dioxoles; D007328:Insulin; D046911:Macromolecular Substances; C076126:disodium (R,R)-5-(2-((2-(3-chlorophenyl)-2-hydroxyethyl)-amino)propyl)-1,3-benzodioxole-2,3-dicarboxylate; D017868:Cyclic AMP-Dependent Protein Kinases; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; C517293:Pde3b protein, mouse