Previously, we have reported that ~ 35 % of rodent adipocyte membrane-associated PDE3B was localized in caveolae-enriched PM, and ~ 65 % in internal membrane (Golgi/ER) fractions [17
]. Our electron microscopy studies indicate that, in human primary adipocytes, PDE3B is also associated with PM/caveolae regions. A very important observation in the present study indicates that activation of distinct membrane pools of PDE3B by insulin or CL in 3T3-L1 adipocytes reflects formation of different macromolecular complexes, i.e. HMWC-ins or HMWC-CL. Before exposure of adipocytes to insulin or CL, PDE3B exhibited an apparent molecular mass of ~ 600–900 kDa during size-exclusion chromatography; after phosphorylation/activation by insulin or CL, the apparent molecular mass of approx. 50 % of the total PDE3B activity increased and eluted at ≥3000 kDa, indicating that phosphorylated PDE3B was recruited into HMWC-ins or HMWC-CL. PDE3B not recruited into HMWC-ins or HMWC-CL, or PDE3B from adipocytes not treated with insulin or CL, exhibited little or no phosphorylation. In addition to phosphorylated PDE3B, HMWC-ins included IRS-1, PI3K p85, PKB/pPKB, HSP-90, 14-3-3, PP2A and cav-1, all of which co-eluted and co-immunoprecipitated with PDE3B after stimulation of adipocytes with insulin. HMWC-CL contained, in addition to phosphorylated/activated PDE3B, several signalling molecules that are probably involved in activation of PDE3B via PKA-mediated signalling pathways. HMWC-CL included PKA-RII, 14-3-3, β
3-AR and cav-1, all of which co-eluted and co-immunoprecipitated with PDE3B after stimulation of adipocytes with CL. HMWC-CL apparently lack IRS-1, PI3K p85, PKB and HSP-90, which were present in HMWC-ins. Molecules common to both HMWC-ins and HMWC-CL included 14-3-3, PP2A, cav-1 and cholesterol. Thus differential phosphorylation/activation of different pools of membrane-associated PDE3B most probably reflects formation of different macromolecular complexes. The consequences of differential regulation of PDE3B at different intracellular locations on the spatial and temporal regulation of cAMP pools and biological responses remain to be elucidated.
PM caveolae are a specialized subset of membrane lipid rafts which are enriched in specific lipids, especially cholesterol and glycosphingolipids, and scaffolding proteins, e.g. caveolins, that interact with numerous proteins and serve as organizers of, or platforms for, diverse signalling pathways [22
]. Recent findings in rodent cardiac myocytes, for example, suggest that bradykinin protects the heart from ischaemia/reperfusion injury by inducing formation of, and internalization of its receptor into, caveolar vesicular signalling complexes, called signalosomes. These signalosomes recruit additional signalling molecules, migrate to mitochondria, and induce opening of the mitoKATP
channel, resulting in cardioprotection [27
]. In adipocytes, caveolae have been implicated in compartmentalization and regulation of cAMP- and insulin-regulated signalling pathways, e.g. lipolysis, lipogenesis and glucose uptake [13
]. Although caveolae represent highly ‘ordered’ lipid rafts with decreased fluidity and greater buoyancy than other PM domains, caveolin also associates, in adipocytes, with other membrane lipid rafts with higher densities, different molar ratios of cholesterol to phospholipid, and different protein compositions, suggesting differential functional segregation in these caveolar or lipid raft subclasses [31
]. Although caveolae are generally resistant to solubilization by non-ionic detergents, changes in glycosphingolipid content, especially ganglioside GM3
, can increase solubilization of membrane-associated cav-1 by non-ionic detergents [32
]. We cannot be certain whether this accounts for solubilization of some membrane-associated cav-1 with 1 % Nonidet P40 in our experiments. It is clear, however, that solubilized cav-1 co-elutes with low-molecular-mass material from control cells; after exposure of 3T3-L1 adipocytes to insulin or CL, some (not all) solubilized cav-1 co-elutes with the HMWCs.
The presence of cav-1 and cholesterol in HMWC-ins or HMWC-CL led us to evaluate the role of cav-1 in regulation of PDE3B. Cav-1−/−
] have allowed the study of cav-1 functions in vivo
. siRNA-mediated down-regulation of CAV-1
in 3T3-L1 adipocytes provided an alternative approach, avoiding possible compensatory mechanisms developed in Cav-1−/−
mice. These two models provide complementary information about the effects of short- and long-term down-regulation of CAV-1
in adipocytes. The results of the present study indicate that siRNA-mediated KD of CAV-1
in 3T3-L1 adipocytes led to reduced expression of PDE3B, perhaps due to increased degradation of PDE3B protein [23
]. Furthermore, in these cells, insulin-induced activation of PDE3B was partially inhibited, whereas CL-induced activation was completely inhibited. Similar results were obtained in primary adipocytes from Cav-1−/−
mice. In CAV-1
-KD adipocytes, CL-induced activation/recruitment of PDE3B was virtually completely blocked, whereas, in the presence of insulin, PDE3 was partially activated and recruited into modified HMWCs (smaller than HMWC-ins, but larger than in control cells).
Activation of lipolysis involves production of cAMP, activation of PKA and translocation of HSL to lipid droplets, elicited by phosphorylated perilipin [35
]. Previous reports suggested that ligand-induced lipolysis in adipocytes may be mediated by association (assessed by co-immunoprecipitation) between cav-1, PKA and perilipin [25
], which facilitates PKA-induced phosphorylation of perilipin. In adipocytes from Cav-1−/−
mice, ligand-induced formation of the complex of PKA, perilipin and cav-1 was prevented, and CL-stimulated lipolysis was inhibited [25
]. Thus reduced phosphorylation of perilipin and, perhaps, of HSL, and reduced lipolysis in CAV-1
-KD adipocytes could be due to defects in the cav-1-mediated association between perilipin and PKA. We suggest that PDE3B may associate/interact with this complex, and disruption of the complex due to down-regulation of cav-1 might prevent CL-induced phosphorylation/activation of PDE3B, as well as of perilipin and HSL. In CAV-1
-KD cells, even if down-regulation of PDE3B expression resulted in increased cAMP, disruption of the PKA–perilipin complex would limit access of PKA to its substrates and inhibit phosphorylation of PKA effectors and cAMP/PKA signalling. With regard to the anti-lipolytic action of insulin, our studies suggest fewer changes in insulin-induced phosphorylation/activation of PKB in CAV-1
-KD adipocytes, where insulin, presumably via PKB, induces partial phosphorylation/activation of PDE3B and its recruitment into modified HMWCs, and inhibits residual CL-stimulated lipolysis. Thus cav-1 may be more important for regulation of cAMP/PKA-mediated activation of PDE3B than for insulin/PKB-mediated activation, in addition to its importance in stabilization of PDE3B expression, and its function as a scaffold for insulin- and CL-induced activation of PDE3B and recruitment of signalling molecules into HMWC-ins and HMWC-CL.
Our previous studies in 3T3-L1 adipocytes suggested that siRNA-induced PDE3B KD did not prevent insulin-stimulated recruitment of signalling molecules into HMWC-ins, indicating that PDE3B is not necessary for HMWC-ins [17
]. In adipocytes, however, recruitment of recombinant PDE3B into HMWC-ins did require the presence of its NT regulatory region [17
], which, in another study, was shown to contain sites phosphorylated in recombinant PDE3B in adipocytes and hepatoma cells stimulated by insulin and cAMP-elevating agents [36
]. In CAV-1
-KD adipocytes, CL-induced phosphorylation/activation/recruitment of PDE3B was virtually completely blocked. In the presence of insulin, however, PDE3B was partially phosphorylated/activated and recruited into modified HMWCs. Wortmannin inhibited insulin-induced phosphorylation/activation of PDE3B and formation of macromolecular complexes [17
]. Thus, although in virtually all of our studies phosphorylation/activation of PDE3B is associated with its recruitment into macromolecular complexes, we cannot be certain whether phosphorylation is required for recruitment, or whether recruitment of PDE3B into (or its association with) macromolecular complexes is required for effective phosphorylation of PDE3B. Future studies with phosphorylation-site mutants in recombinant PDE3B, expressed in adipocytes, may shed light on the overall relationship, if any, between signalling and complex formation, as well as the role of phosphorylation of PDE3B in its activation and/or recruitment into HMWCs.
Taken together, these studies provide an important example, in a single cell, for subcellular localization of PDE3B, cav-1 and other signalling molecules, and their roles in the differential regulation of membrane-associated PDE3B in different subcellular compartments, via PKA and PKB signalling pathways. Different PDE4D variants, i.e. PDE4D3 and PDE4D8, have been reported to co-immunoprecipitate with PKA–AKAP (PKA-anchoring protein) complexes in different cells, i.e. cardiac and vascular myocytes respectively [37
]. The present study also demonstrates the importance of membrane integrity in the formation/maintenance of insulin- and CL-induced macromolecular assemblies that contain PDE3B, and suggests a chaperone or scaffolding role of cav-1 in cholesterol-rich lipid domains that may be necessary for stabilization and phosphorylation/activation of PDE3B within these macromolecular signalling complexes.