Differential regulation of adipocyte PDE3B in distinct membrane compartments by insulin and the β3-adrenergic receptor agonist CL316243: effects of caveolin-1 knockdown on formation/maintenance of macromolecular signalling complexes

被引:38
作者
Ahmad, Faiyaz [1 ]
Lindh, Rebecka [2 ]
Tang, Yan [1 ]
Ruishalme, Iida [3 ,4 ]
Ost, Anita [3 ,4 ]
Sahachartsiri, Bobby [1 ]
Stralfors, Peter [3 ,4 ]
Degerman, Eva [2 ]
Manganiello, Vincent C. [1 ]
机构
[1] NHLBI, Translat Med Branch, NIH, Bethesda, MD 20892 USA
[2] Lund Univ, Dept Expt Med Sci, S-22184 Lund, Sweden
[3] Linkoping Univ, Div Cell Biol, SE-58185 Linkoping, Sweden
[4] Linkoping Univ, Diabet Res Ctr, SE-58185 Linkoping, Sweden
基金
英国医学研究理事会;
关键词
adipocyte; beta(3)-adrenergic receptor; caveolin-1; insulin; protein kinase A (PKA); phosphodiesterase 3 (PDE3); NUCLEOTIDE PHOSPHODIESTERASE 3B; HORMONE-SENSITIVE LIPASE; PROTEIN-KINASE B; RAT ADIPOCYTES; INDUCED PHOSPHORYLATION; PLASMA-MEMBRANE; ACTIVATION; TRANSLOCATION; CELLS; MICE;
D O I
10.1042/BJ20090842
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:399 / 410
页数:12
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