Homologous or agonist-specific desensitization of beta-adrenergic receptors is thought to be mediated by a specific kinase, the beta-adrenergic receptor kinase (beta ARK). However, recent data suggest that a cofactor is required for this kinase to inhibit receptor function. The complementary DNA for such a cofactor was cloned and found to encode a 418-amino acid protein homologous to the retinal protein arrestin. The protein, termed beta-arrestin, was expressed and partially purified. It inhibited the signaling function of beta ARK-phosphorylated beta-adrenergic receptors by more than 75 percent, but not that of rhodopsin. It is proposed that beta-arrestin in concert with beta ARK effects homologous desensitization of beta-adrenergic receptors.

The current view of how steroid hormone receptors affect gene transcription is that these receptors, on binding ligand, change to a state in which they can interact with chromatin and regulate transcription of target genes. Receptor activation is believed to be dependent only on this ligand-binding event. Selected steroid hormone receptors can be activated in a ligand-independent manner by a membrane receptor agonist, the neurotransmitter dopamine. In vitro, dopamine faithfully mimicked the effect of progesterone by causing a translocation of chicken progesterone receptor (cPR) from cytoplasm to nucleus. Dual activation by progesterone and dopamine was dissociable, and a serine residue in the cPR was identified that is not necessary for progesterone-dependent activation of cPR, but is essential for dopamine activation of this receptor.

The (3-adrenergic receptor kinase is an enzyme, possibly analogous to rhodopsin kinase, that multiply phosphorylates the (3-adrenergic receptor only when it is occupied by stimulatory agonists. Since this kinase may play an important role in mediating the process of homologous, or agonist-specific, desensitization, we investigated the functional consequences of receptor phosphorylation by the kinase and possible analogies with the mechanism of action of rhodopsin kinase. Pure hamster lung 182-adrenergic receptor, reconstituted in phospholipid vesicles, was assessed for its ability to mediate agonist-promoted stimulation of the GTPase activity of coreconstituted stimulatory guanine nucleotide-binding regulatory protein. When the receptor was phosphorylated by partially ("350-fold) purified preparations of ,B-adrenergic receptor kinase, as much as 80% inactivation of its functional activity was observed. However, the use of more highly purified enzyme preparations led to a dramatic decrease in the ability of phosphorylation to inactivate the receptor such that pure enzyme preparations (""20,000-fold purified) caused only minimal (""16 ± 7%) inactivation. Addition of pure retinal arrestin (48-kDa protein or S antigen), which is involved in enhancing the inactivating effect of rhodopsin phosphorylation by rhodopsin kinase, led to partial restoration of the functional effect of ,B-adrenergic receptor kinase-promoted phosphorylation (41 + 3% inactivation). These results suggest the possibility that a protein analogous to retinal arrestin may exist in other tissues and function in concert with ,I-adrenergic receptor kinase to regulate the activity of adenylate cyclase-coupled receptors.Transmembrane signaling systems for converting extracellular stimuli as divergent as hormones, drugs, or photons of light into intracellular metabolic changes have been remarkably conserved through evolution. Such systems generally consist of three major components: a receptor, such as the (6).In the present studies we have investigated the functional consequences of phosphorylation of purified mammalian f82-adrenergic receptor with ,AR kinase by assessing the ability of these receptors to mediate the agonist-promoted stimulation of the GTPase activity of Gs in a reconstituted system. Moreover, a functionally important interaction of purified retinal arrestin with this system is documented. METHODSProtein Purification. ,B-Adrenergic receptor from hamster lung was purified to >95% homogeneity by sequential affinity chromatography and HPLC as described (10).Gs was purified from a cholate extract of washed human erythrocyte membranes as described (11). The preparations used in these experiments were from step 8A in ref. 11 and were >95% pure as judged by Coomassie blue staining of polyacrylamide gels.,BAR kinase was purified from bovine brain by (NH4)2SO4 precipitation of a high-speed supernatant fraction as described (12). The precipitate was then chromatographed on Ultrogel AcA 34, DEAE-Sephacel, CM-Fractogel, and hydroxylapatite...