Differential regulation of adipocyte PDE3B in distinct membrane compartments by insulin and the beta(3)-adrenergic receptor agonist CL316243: effects of caveolin-1 knockdown on formation/maintenance of macromolecular signalling complexes
2009 (English)In: BIOCHEMICAL JOURNAL, ISSN 0264-6021, Vol. 424, no 3, 399-410 p.Article in journal (Refereed) Published
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 beta(3)-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 KID 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 P-32-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 beta(3)-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 KID. 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.
Place, publisher, year, edition, pages
2009. Vol. 424, no 3, 399-410 p.
adipocyte, beta(3)-adrenergic receptor, caveolin-1, insulin, protein kinase A (PKA), phosphodiesterase 3 (PDE3)
National CategoryMedical and Health Sciences
IdentifiersURN: urn:nbn:se:liu:diva-53067DOI: 10.1042/BJ20090842OAI: oai:DiVA.org:liu-53067DiVA: diva2:286633