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

Abstract

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 β3-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 β3-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.

The Role of PDE3B Phosphorylation in the Inhibition of Lipolysis by Insulin

“…Localized regulation of PKA activity by the assembly of signaling complexes containing components of the cAMP/PKA pathway is a recurrent theme in cellular physiology, and such a mechanism seems plausible for the regulation of lipolysis at the lipid droplet (53)(54)(55). In support of this hypothesis, in primary and 3T3-L1 adipocytes, PDE3B associates with caveolin-1, a scaffolding protein within plasma membrane caveolae, which localizes to the lipid droplet upon ␤-adrenergic stimulation (56)(57)(58)(59)(60). Additionally, in the current study, we show that endogenous PDE3B localizes to the lipid droplet fraction of brown adipocytes, suggesting that this compartment might be a site of PDE3B action.…”

Mammalian Cyclic Nucleotide Phosphodiesterases: Molecular Mechanisms and Physiological Functions

“…Identities in amino acid sequences among C domains range from 25-51%, but high sequence identity does not translate into functional similarities. PDEs 5,6, and 11 are among the most similar in amino acid sequence, but their substrate preferences, catalytic rates, and substrate affinities vary markedly (30,70,282). Moreover, substrate preference does not translate into structural similarities; the x-ray crystallographic structure of PDE9, a cGMP-specific PDE, is more like that of the PDE4 family members, which are cAMP-specific PDEs, than that of other cGMP-specific PDEs (163).…”

“…PDE localization to subcellular compartments can occur by various mechanisms including recruitment to lipid rafts, AKAPs, or other anchoring proteins like ␤-arrestin (6,95,340,374). Indeed, members of the same PDE family located in different regions of the cell have been shown to be selectively modulated by actions of single or different agonists (6,410).…”

“…Evidence suggests that creation of cAMP microdomains requires fastidiously compartmentalized and anchored pools of PDEs that tightly regulate cN action at specific points of interest in the cell, and cAMP-hydrolyzing PDEs that have different affinities for cAMP within the same cell may be involved in physiological responses at different levels of intracellular cAMP (6,96,391). PDE localization to subcellular compartments can occur by various mechanisms including recruitment to lipid rafts, AKAPs, or other anchoring proteins like ␤-arrestin (6,95,340,374). Indeed, members of the same PDE family located in different regions of the cell have been shown to be selectively modulated by actions of single or different agonists (6,410).…”

“…Despite widespread acceptance that magnesium is required for PDE catalytic function, this is not a certainty since for most PDEs other metals support activity equally FIG. 6. Depiction of position of cAMP and cGMP in the catalytic site of PDE 10A2.…”