The β-Adrenergic Receptor Kinase: Role in Homologous Desensitization in S49 Lymphoma Cells

Summary

Phosphorylation of the β-adrenergic receptor (BAR) is closely associated with homologous desensitization of the B-adrenergic receptor-coupled adenylate cyclase system. Homologous desensitization and receptor phosphorylation also occur in cell mutants which are deficient in their cAMP-dependent protein kinase (kin^- mutant of S49 lymphoma cells). βAR phosphorylation is mediated by a cAMP-independent protein kinase which phosphorylates the receptor only when it is occupied by a B-agonist. During the time course of desensitization the BAR kinase (βARK) activity is translocated from a cytoplasmic to a plasma membrane location. βARK translocation can also be effected by prostaglandin E₁ (PGE₁) suggesting that this βARK may represent a more general enzyme capable of phosphorylating other adenylate cyclase-coupled receptors. Thus, BARK may play a key role in the process of homologous desensitization of adenylate cyclase coupled receptors.

Extracellular hormones interact with specific receptors at the outer surface of the plasma membrane and thus initiate a cellular response. One of the best studied transmembrane signalling systems known to be coupled to the occupancy of cell surface receptors is adenylate cyclase. The adenylate cyclase system is composed of various components all of which have been purified to homogeneity (Shorr et al., 1982; Homcy et al., 1983; Benovic et al., 1984; Codina et al., 1984; Northup et al., 1980; Sternweis et al., 1981; Bokoch et al., 1984; Pfeuffer et al., 1985). Initially, agonist binding to the receptor promotes coupling of the occupied receptor to one of the guanine nucleotide binding regulatory proteins. These proteins are members of a family of heterotrimeric proteins consisting of α, β and γ subunits. Stimulatory receptors like the β-adrenergic (Cerione et al., 1984) or glucagon (Iyengar et al., 1979) receptors couple to the stimulatory regulatory protein N_s (or G_s) whereas inhibitory receptors like the α₂-adrenergic (Jacobs et al., 1976) or M₂-muscarinic (Harden et al., 1982) receptors couple to the inhibitory regulatory protein N_i (or G_i).

Prolonged exposure to agonist hormones, either stimulatory or inhibitory, results in an attenuation of the response to the hormonal activation, a phenomenon called tachyphylaxis or desensitization (Harden, 1983; Sibley and Lefkowitz, 1985; Sharma et al., 1975). One of the best studied models for desensitization is the β-adrenergic receptor-coupled adenylate cyclase system. In this system two different forms of desensitization have been characterized. Homologous or hormone-specific desensitization results in an attenuated response only to the desensitizing hormone. In contrast, the heterologous form of desensitization leads to a general decrease of adenylate cyclase activity promoted not only by the desensitizing hormone but by other hormones and non-hormonal stimulators as well.

Previous studies have demonstrated that phosphorylation of the β-adrenergic receptor is involved in the mechanism of heterologous desensitization (Stadel et al., 1983; Sibley et al., 1984). In this form of desensitization phosphorylation of the β-adrenergic receptor is at least in part cAMP-dependent and mediated by the cAMP-dependent protein kinase (protein kinase A) (Strulovici et al., 1984; Sibley et al., 1984; Benovic et al., 1985).

Homologous desensitization, however, appears to be independent of cAMP since it has been observed in systems which are defective in their cAMP-dependent pathway (Green and Clark, 1981; Green et al., 1981; Perkins, 1983; Clark et al., 1985). These systems either lack the N protein or a functional cAMP-dependent protein kinase. Consequently β-adrenergic receptor occupancy does not result in an increase in intracellular cAMP levels (cyc^- mutant of S49 lymphoma cells) (Bourne et al., 1975; Bourne et al., 1981; Ross and Gilman, 1977) or cAMP-dependent protein phosphorylation (kin^- mutant of S49 lymphoma cells) (Steer et al., 1976; Steinberg et al., 1978; Mahan et al., 1985). Therefore, if phosphorylation of the β-adrenergic receptor is involved in the process of homologous desensitization it must be catalyzed by a non cAMP-dependent protein kinase. To address these questions we utilized the kin mutant of the S49 lymphoma cells (Steer et al., 1976; Steinberg et al., 1978; Mahan et al., 1985). We document here a cAMP independent pathway of β-adrenergic receptor active phosphorylation during homologous desensitization. The kinase involved in this phosphorylation process is distinct from other known kinases and phosphorylates only the agonist occupied form of the β-adrenergic receptor. Moreover, during desensitization the cytosolic kinase activity becomes transiently translocated to the plasma membranes in a cAMP-independent manner.