Dynamic Complexes of β2-Adrenergic Receptors with Protein Kinases and Phosphatases and the Role of Gravin

Abstract

Signals mediated by G-protein-linked receptors display agonist-induced attenuation and recovery involving both protein kinases and phosphatases. The role of protein kinases and phosphatases in agonist-induced attenuation and recovery of ␤-adrenergic receptors was explored by two complementary approaches, antisense RNA suppression and co-immunoprecipitation of target elements. Protein phosphatases 2A and 2B are associated with the unstimulated receptor, the latter displaying a transient decrease followed by a 2-fold increase in the levels of association at 30 min following challenge with agonist. Protein kinase A displays a robust, agonist-induced association with ␤-adrenergic receptors over the same period. Suppression of phosphatases 2A and 2B with antisense RNA or inhibition of their activity with calyculin A and FK506, respectively, blocks resensitization following agonist removal. Recycling of receptors to the plasma membrane following agonist-promoted sequestration is severely impaired by loss of either phosphatase 2B or protein kinase C. In addition, loss of protein kinase C diminishes association of phosphatase 2B with ␤-adrenergic receptors. Overlay assays performed with the RII subunit of protein kinase A and co-immunoprecipitations reveal proteins of the A kinase-anchoring proteins (AKAP) family, including AKAP250 also known as gravin, associated with the ␤-adrenergic receptor. Suppression of gravin expression disrupts recovery from agonist-induced desensitization, confirming the role of gravin in organization of G-protein-linked signaling complexes. The Ht31 peptide, which blocks AKAP protein-protein interactions, blocks association of ␤-adrenergic receptors with protein kinase A. These data are the first to reveal dynamic complexes of ␤-adrenergic receptors with protein kinases and phosphatases acting via an anchoring protein, gravin.

AKAPs (A-kinase anchoring proteins) and molecules that compose their G-protein-coupled receptor signalling complexes

“…The multivalency of AKAP250 has since increased with discovery of its association with a GPCR {the β 2 -adrenergic receptor (β 2 AR) [17][18][19]22,23]}, the non-receptor tyrosine kinase Src [24,25] and other proteins such as CaN (see below). Although for more than a decade CaN, PKA and PKC were known to be expressed in post-synaptic densities and to function in neural plasticity, only in the last several years has the pivotal role of AKAPs in providing a scaffold for these signalling elements been revealed.…”

“…The protein phosphatase CaN has been shown to be associated with AKAP79 [12] and with β 2 AR-based signalling complexes [19,[40][41][42]. CaN associates with either AKAP, and appears to be 'silenced' (i.e.…”

“…CaN associates with either AKAP, and appears to be 'silenced' (i.e. enzymic activity suppressed) through binding, rather than simply tethered to the scaffold molecules [12,40]. Recognition that AKAP association with CaN is able to suppress phosphatase activity expanded our understanding of the function of AKAPs.…”

“…The peptide comprising residues 330-357 is 50-fold more potent, however, than the peptide of AKAP79 comprising residues 88-101 [43]. Although association with the β 2 AR-based signalling complex clearly silences CaN [40], the binding sites for CaN on AKAP250 have not been mapped. There are no regions in AKAP250 with high similarity to either the dominant (residues 330-357) or lesser (residues 88-101) sequences of AKAP79.…”

“…The demonstration that both AKAP79 [47] and AKAP250 [40] scaffolds bind to the β 2 AR, a prototypical GPCR [44], achieved another important milestone in our understanding of the roles of AKAPs in multivalent signalling complexes [44]. Intuitively, one can appreciate why there are spatial advantages provided by an AKAP scaffold, localizing PKA, PKC and CaN in close proximity to a GPCR that couples downstream to cAMP production.…”