A Key Role for Protein Kinase A in Homologous Desensitization of the β2-Adrenergic Receptor Pathway in S49 Lymphoma Cells

Abstract

Desensitization of receptor-mediated cellular responses is a complex process which can involve multiple pathways including: (a) receptor uncoupling from G protein, (b) sequestration of receptors away from the plasma membrane, and (c) downregulation of signaling proteins (1-4). It has been demonstrated that uncoupling of receptor and G protein occurs rapidly (within 10 min) in response to receptor activation and appears to involve receptor phosphorylation (5). In the case of the ␤ 2 -adrenergic receptor (␤ 2 -AR) 1 system, agonist-occupied receptors activate the stimulatory G protein, G s , which in turn promotes the production of cAMP by adenylyl cyclase. Increased cAMP levels activate the cAMP-dependent protein kinase (PKA) resulting in the phosphorylation of several proteins, including the ␤ 2 -AR and other receptors. In addition, agonist-occupied ␤ 2 -AR are phosphorylated by one or more isoforms of the ␤-adrenergic receptor kinase (␤-ARK, also termed G protein receptor kinase; GRK) resulting in the uncoupling of receptor and G s (6 -10). Therefore, both PKA and ␤-ARK can contribute to desensitization of the ␤ 2 -AR system. In contrast to the effect of ␤-ARK, PKA-mediated ␤ 2 -AR phosphorylation is most often associated with heterologous desensitization (that which is agonist nonspecific and therefore not dependent on ␤ 2 -AR occupancy), but is generally thought to be of little importance in homologous desensitization (agonistspecific and dependent on ␤ 2 -AR occupancy) (11-14). Therefore, while the ␤ 2 -AR can be phosphorylated by both PKA and ␤-ARK, most recent work has focused on the importance of ␤-ARK and related proteins (reviewed in Refs. 6 and 15-17) as mediators of homologous desensitization.The kin Ϫ S49 murine lymphoma cell lacks PKA activity and thus provides a unique system in which to study the relative importance of PKA in mediating various cellular responses. Importantly, both WT and kin Ϫ S49 cells express only the ␤ 2 -AR isoform and display similar profiles of ␤-AR agonistinduced desensitization of cAMP production and cell-surface ␤ 2 -AR loss (14, 18). The ability of the cell-permeable cAMP analog, 8-Br-cAMP, to mimic the effect of epinephrine in decreasing ␤-AR agonist-induced adenylyl cyclase activity in membranes prepared from treated cells suggest that the effect of PKA activation is not dependent on the presence of agonist (11). These results suggest that PKA is not involved in homologous desensitization of the ␤ 2 -AR.In the current studies, we have used a [ 3 H]forskolin binding assay to define more precisely the role of PKA in desensitizing the ␤ 2 -AR system in intact S49 cells. Since high-affinity forskolin interaction with adenylyl cyclase requires the activation of G␣ s , the amount of [ 3 H]forskolin associated with cells can be used as an index of G␣ s -adenylyl cyclase interaction (19 -21). This assay, therefore, both provides a direct means of assessing hormone-stimulated G␣ s -adenylyl cyclase interaction in intact cells that is independent of second messenger accumulation...

Regulation of Dopamine D1Receptor Trafficking by Protein Kinase A-Dependent Phosphorylation

“…For ␤-adrenergic receptors, a distinction has been made between desensitization that is homologous (resulting from stimulation of the same receptor) or heterologous (resulting from stimulation of a different receptor subtype, or nonreceptor-mediated activation of a second messenger) (Harden, 1983). A mechanism for heterologous desensitization is phosphorylation of the receptor by PKA (Clark et al, 1989;Hausdorff et al, 1989), whereas homologous desensitization of the ␤ 2 -adrenergic receptor involves phosphorylation of the agonist-occupied receptor by both GRK (Benovic et al, 1986) and PKA (Post et al, 1996;Moffett et al, 2001). Heterologous desensitization of the ␤ 2 -adrenergic receptor is mediated by PKA-dependent phosphorylation of Ser262, located in the carboxyl terminal portion of the third cytoplasmic loop, a position similar to that of Thr268 in the D 1 receptor (Clark et al, 1989;Hausdorff et al, 1989;Yuan et al, 1994).…”

Regulatory mechanisms that modulate signalling by G-protein-coupled receptors

“…PKA induces phosphorylation and heterologous desensitization of the β # -AR [116][117][118]. Desensitization of the responses of S49 lymphoma cells to isoprenaline are markedly reduced in kin − cells, which lack PKA, indicating that PKA also mediates homologous desensitization of the β # -AR [119]. Like the GRKs, PKA is also translocated from the cytosol to a particulate fraction after agonist exposure [92].…”

Reduction of G Protein-Coupled Receptor Kinase 2 Expression in U-937 Cells Attenuates H2 Histamine Receptor Desensitization and Induces Cell Maturation

“…G protein-coupled receptor kinases (GRKs) are responsible for homologous desensitization, whereas the second messenger-dependent kinases, protein kinases A (PKA) and C, could be involved in homologous and heterologous desensitization (Freedman and Lefkowitz, 1996;Post et al, 1996;Moffett et al, 2001). GRKmediated phosphorylation of serine/threonine residues in the carboxyl tail and/or intracellular loops of GPCRs increases the receptor affinity to arrestin-type proteins, and this binding prevents any further coupling between the receptor and This study was supported by grants from the University of Buenos Aires (grant BO22), the Consejo Nacional de Investigaciones Científicas y Tecnológi-cas, (PID 792/98), the Agencia Nacional de Promoción Científica y Técnológica, , and the Ministerio de Salud de la Nación Argentina (Carrillo-Oñ ativia grant).…”