Regulation of tyrosine kinase cascades by G-protein-coupled receptors.

Mitogenic signaling by G-protein-coupled receptors (GPCRs) involves tyrosine phosphorylation of adaptor proteins and assembly of multiprotein Ras activation complexes. Over the past three years, three types of scaffolds for GPCR-directed complex assembly have been identified: transactivated receptor tyrosine kinases (RTKs), integrin-based focal adhesions, and GPCRs themselves. Nonreceptor tyrosine kinases play an important role in each case. The processes of GPCR desensitization and sequestration via clathrin-coated pits are also involved in signaling through the RTK- and GPCR-based scaffolds.

Expanding roles for beta-arrestins as scaffolds and adapters in GPCR signaling and trafficking.

beta-arrestins play previously unsuspected and important roles as adapters and scaffolds that localize signaling proteins to ligand-activated G-protein-coupled receptors. As with the paradigmatic role of the beta-arrestins in uncoupling receptors from G proteins (desensitization), these novel functions involve the interaction of beta-arrestin with phosphorylated heptahelical receptors. beta-arrestins interact with at least two main classes of signaling proteins. First, interaction with molecules such as clathrin, AP-2 and NSF directs the clathrin-mediated internalization of G-protein-coupled receptors. Second, interaction with molecules such as Src, Raf, Erk, ASK1 and JNK3 appears to regulate several pathways that result in the activation of MAP kinases. These recent discoveries indicate that the beta-arrestins play widespread roles as scaffolds and/or adapter molecules that organize a variety of complex signaling pathways emanating from heptahelical receptors. It is likely that additional roles for the beta-arrestins remain to be discovered.

The superfamily of heptahelical receptors.

Despite a growing appreciation of functional analogies between visual and hormonal signalling systems in the early 1980s, the discovery of the close structural relationship between rhodopsin and the beta2-adrenergic receptor, and of the existence of a larger 'superfamily' of such receptors, came as a total surprise. Here I provide a personal perspective on events leading up to and flowing from this exciting discovery that opened up a vast field of research.

Alpha-adrenergic receptor identification by (3H)dihydroergocryptine binding.

A radioactively labeled alpha-adrenergic antagonist, [3H]dihydroergocryptine, binds specifically to a site on rabbit uterine membranes. Binding is rapid, reaching equilibrium in less than 17 minutes at 25 degrees C. Adrenergic agonists compete for this binding site with an order of affinities identical to the pharmacological potency order of these agents as alpha-adrenergic agonists (epinephrine greater than norepinephrine greater than isoprotereonl). The (-) stereoisomers of epinephrine and norepinephrine are 30 times more potent in competing for the site than the corresponding (+) stereoisomers. alpha-Adrenergic antagonists, such as phentolamine and phenoxybenzamine, potently compete for the binding sites while the beta-adrenergic antagonist propranolol does not. Structural analogs of catecholamines that are devoid of alpha-adrenergic physiological activity do not compete for [3H]dihydroergocryptine binding sites. These data suggest that alpha-adrenergic receptors can be directly identified and studied by [3H]dihydroergocryptine binding.

Radioreceptor assay of adrenocorticotropic hormone: new approach to assay of polypeptide hormones in plasma.

Biologically active iodine-125-labeled adrenocorticotropic hormone (ACTH) binds specifically to ACTH receptors extracted from adrenals. Unlabeled ACTH at 1 picogram per milliliter significantly displaces labeled ACTH from these receptors. This system, which appears to be applicable to all polypeptide hormones, provides a rapid and sensitive method for measurements of biologically active ACTH in dilute whole plasma.

Antagonism of catecholamine receptor signaling by expression of cytoplasmic domains of the receptors.

The actions of many hormones and neurotransmitters are mediated by the members of a superfamily of receptors coupled to heterotrimeric guanine nucleotide-binding proteins (G proteins). These receptors are characterized by a highly conserved topographical arrangement in which seven transmembrane domains are connected by intracellular and extracellular loops. The interaction between these receptors and G proteins is mediated in large part by the third intracellular loop of the receptor. Coexpression of the third intracellular loop of the alpha 1B-adrenergic receptor with its parent receptor inhibited receptor-mediated activation of phospholipase C. The inhibition extended to the closely related alpha 1C-adrenergic receptor subtype, but not the phospholipase C-coupled M1 muscarinic acetylcholine receptor nor the adenylate cyclase-coupled D1A dopamine receptor. These results suggest that the receptor-G protein interface may represent a target for receptor antagonist drugs.

Beta-adrenergic receptor kinase: primary structure delineates a multigene family.

The beta-adrenergic receptor kinase (beta-ARK), which specifically phosphorylates only the agonist-occupied form of the beta-adrenergic and closely related receptors, appears to be important in mediating rapid agonist-specific (homologous) desensitization. The structure of this enzyme was elucidated by isolating clones from a bovine brain complementary DNA library through the use of oligonucleotide probes derived from partial amino acid sequence. The beta-ARK cDNA codes for a protein of 689 amino acids (79.7 kilodaltons) with a protein kinase catalytic domain that bears greatest sequence similarity to protein kinase C and the cyclic adenosine monophosphate (cyclic AMP)--dependent protein kinase. When this clone was inserted into a mammalian expression vector and transfected into COS-7 cells, a protein that specifically phosphorylated the agonist-occupied form of the beta 2-adrenergic receptor and phosphorylated, much more weakly, the light-bleached form of rhodopsin was expressed. RNA blot analysis revealed a messenger RNA of four kilobases with highest amounts in brain and spleen. Genomic DNA blot analysis also suggests that beta-ARK may be the first sequenced member of a multigene family of receptor kinases.

Direct demonstration of impaired functionality of a purified desensitized beta-adrenergic receptor in a reconstituted system.

Long-term exposure of various cell types to beta-adrenergic agonists such as isoproterenol leads to an attenuated responsiveness ("desensitization") of the adenylate cyclase system to further challenge with these agonists. The turkey erythrocyte model system was used earlier to show that a covalent modification of the receptor (phosphorylation) is associated with this process. The functionality of the "desensitized" beta-adrenergic receptor was assessed by implanting purified beta-adrenergic receptor preparations from control and desensitized turkey erythrocytes into phospholipid mixtures and then fusing them with receptor-deficient cells (Xenopus laevis erythrocytes). Desensitized beta-adrenergic receptors showed a 40 to 50 percent reduction in their ability to couple to the heterologous adenylate cyclase system, comparable to the reduction in their functionality observed in their original membrane environment. These results demonstrate the utility of recently developed receptor reconstitution techniques for assessing the functionality of purified receptors and show a direct link between a covalent modification of a membrane-bound receptor and its impaired functionality in a reconstituted system.

Control of yeast mating signal transduction by a mammalian beta 2-adrenergic receptor and Gs alpha subunit.

To facilitate functional and mechanistic studies of receptor-G protein interactions, [corrected] the human beta 2-adrenergic receptor (h beta-AR) has been expressed in Saccharomyces cerevisiae. This was achieved by placing a modified h beta-AR gene under control of the galactose-inducible GAL1 promoter. After induction by galactose, functional h beta-AR was expressed at a concentration several hundred times as great as that found in any human tissue. As determined from competitive ligand binding experiments, h beta-AR expressed in yeast displayed characteristic affinities, specificity, and stereoselectivity. Partial activation of the yeast pheromone response pathway by beta-adrenergic receptor agonists was achieved in cells coexpressing h beta-AR and a mammalian G protein (Gs) alpha subunit-demonstrating that these components can couple to each other and to downstream effectors when expressed in yeast. This in vivo reconstitution system provides a new approach for examining ligand binding and G protein coupling to cell surface receptors.

beta-Arrestin: a protein that regulates beta-adrenergic receptor function.

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.

Regulation of receptor fate by ubiquitination of activated beta 2-adrenergic receptor and beta-arrestin.

Although trafficking and degradation of several membrane proteins are regulated by ubiquitination catalyzed by E3 ubiquitin ligases, there has been little evidence connecting ubiquitination with regulation of mammalian G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR) function. Agonist stimulation of endogenous or transfected beta2-adrenergic receptors (beta2ARs) led to rapid ubiquitination of both the receptors and the receptor regulatory protein, beta-arrestin. Moreover, proteasome inhibitors reduced receptor internalization and degradation, thus implicating a role for the ubiquitination machinery in the trafficking of the beta2AR. Receptor ubiquitination required beta-arrestin, which bound to the E3 ubiquitin ligase Mdm2. Abrogation of beta-arrestin ubiquitination, either by expression in Mdm2-null cells or by dominant-negative forms of Mdm2 lacking E3 ligase activity, inhibited receptor internalization with marginal effects on receptor degradation. However, a beta2AR mutant lacking lysine residues, which was not ubiquitinated, was internalized normally but was degraded ineffectively. These findings delineate an adapter role of beta-arrestin in mediating the ubiquitination of the beta2AR and indicate that ubiquitination of the receptor and of beta-arrestin have distinct and obligatory roles in the trafficking and degradation of this prototypic GPCR.

Cardiac function in mice overexpressing the beta-adrenergic receptor kinase or a beta ARK inhibitor.

Transgenic mice were created with cardiac-specific overexpression of the beta-adrenergic receptor kinase-1 (beta ARK1) or a beta ARK inhibitor. Animals overexpressing beta ARK1 demonstrated attenuation of isoproterenol-stimulated left ventricular contractility in vivo, dampening of myocardial adenylyl cyclase activity, and reduced functional coupling of beta-adrenergic receptors. Conversely, mice expressing the beta ARK inhibitor displayed enhanced cardiac contractility in vivo with or without isoproterenol. These animals demonstrate the important role of beta ARK in modulating in vivo myocardial function. Because increased amounts of beta ARK1 and diminished cardiac beta-adrenergic responsiveness characterize heart failure, these animals may provide experimental models to study the role of beta ARK in heart disease.

Beta-adrenergic receptor kinase-2 and beta-arrestin-2 as mediators of odorant-induced desensitization.

beta-Adrenergic receptor kinase (beta ARK) and beta-arrestin function in the homologous or agonist-activated desensitization of G protein-coupled receptors. The isoforms beta ARK-2 and beta-arrestin-2 are highly enriched in and localized to the dendritic knobs and cilia of the olfactory receptor neurons where the initial events of olfactory signal transduction occur. Odorants induce a rapid and transient elevation of adenosine 3',5'-monophosphate (cAMP), which activates a nonspecific cation channel and produces membrane depolarization. Preincubation of rat olfactory cilia with antibodies raised against beta ARK-2 and beta-arrestin-2 increased the odorant-induced elevation of cAMP and attenuated desensitization. These results suggest that beta ARK-2 and beta-arrestin-2 mediate agonist-dependent desensitization in olfaction.

Chimeric alpha 2-,beta 2-adrenergic receptors: delineation of domains involved in effector coupling and ligand binding specificity.

The alpha 2 and beta 2 adrenergic receptors, both of which are activated by epinephrine, but which can be differentiated by selective drugs, have opposite effects (inhibitory and stimulatory) on the adenylyl cyclase system. The two receptors are homologous with each other, rhodopsin, and other receptors coupled to guanine nucleotide regulatory proteins and they contain seven hydrophobic domains, which may represent transmembrane spanning segments. The function of specific structural domains of these receptors was determined after construction and expression of a series of chimeric alpha 2-,beta 2-adrenergic receptor genes. The specificity for coupling to the stimulatory guanine nucleotide regulatory protein lies within a region extending from the amino terminus of the fifth hydrophobic domain to the carboxyl terminus of the sixth. Major determinants of alpha 2- and beta 2-adrenergic receptor agonist and antagonist ligand binding specificity are contained within the seventh membrane spanning domain. Chimeric receptors should prove useful for elucidating the structural basis of receptor function.

Targeting the receptor-Gq interface to inhibit in vivo pressure overload myocardial hypertrophy.

Hormones and neurotransmitters may mediate common responses through receptors that couple to the same class of heterotrimeric guanine nucleotide-binding (G) protein. For example, several receptors that couple to Gq class proteins can induce cardiomyocyte hypertrophy. Class-specific inhibition of Gq-mediated signaling was produced in the hearts of transgenic mice by targeted expression of a carboxyl-terminal peptide of the alpha subunit Galphaq. When pressure overload was surgically induced, the transgenic mice developed significantly less ventricular hypertrophy than control animals. The data demonstrate the role of myocardial Gq in the initiation of myocardial hypertrophy and indicate a possible strategy for preventing pathophysiological signaling by simultaneously blocking multiple receptors coupled to Gq.

Enhanced morphine analgesia in mice lacking beta-arrestin 2.

The ability of morphine to alleviate pain is mediated through a heterotrimeric guanine nucleotide binding protein (G protein)-coupled heptahelical receptor (GPCR), the mu opioid receptor (muOR). The efficiency of GPCR signaling is tightly regulated and ultimately limited by the coordinated phosphorylation of the receptors by specific GPCR kinases and the subsequent interaction of the phosphorylated receptors with beta-arrestin 1 and beta-arrestin 2. Functional deletion of the beta-arrestin 2 gene in mice resulted in remarkable potentiation and prolongation of the analgesic effect of morphine, suggesting that muOR desensitization was impaired. These results provide evidence in vivo for the physiological importance of beta-arrestin 2 in regulating the function of a specific GPCR, the muOR. Moreover, they suggest that inhibition of beta-arrestin 2 function might lead to enhanced analgesic effectiveness of morphine and provide potential new avenues for the study and treatment of pain, narcotic tolerance, and dependence.

Beta-arrestin 2: a receptor-regulated MAPK scaffold for the activation of JNK3.

beta-Arrestins, originally discovered in the context of heterotrimeric guanine nucleotide binding protein-coupled receptor (GPCR) desensitization, also function in internalization and signaling of these receptors. We identified c-Jun amino-terminal kinase 3 (JNK3) as a binding partner of beta-arrestin 2 using a yeast two-hybrid screen and by coimmunoprecipitation from mouse brain extracts or cotransfected COS-7 cells. The upstream JNK activators apoptosis signal-regulating kinase 1 (ASK1) and mitogen-activated protein kinase (MAPK) kinase 4 were also found in complex with beta-arrestin 2. Cellular transfection of beta-arrestin 2 caused cytosolic retention of JNK3 and enhanced JNK3 phosphorylation stimulated by ASK1. Moreover, stimulation of the angiotensin II type 1A receptor activated JNK3 and triggered the colocalization of beta-arrestin 2 and active JNK3 to intracellular vesicles. Thus, beta-arrestin 2 acts as a scaffold protein, which brings the spatial distribution and activity of this MAPK module under the control of a GPCR.

Cloning, sequencing, and expression of the gene coding for the human platelet alpha 2-adrenergic receptor.

The gene for the human platelet alpha 2-adrenergic receptor has been cloned with oligonucleotides corresponding to the partial amino acid sequence of the purified receptor. The identity of this gene has been confirmed by the binding of alpha 2-adrenergic ligands to the cloned receptor expressed in Xenopus laevis oocytes. The deduced amino acid sequence is most similar to the recently cloned human beta 2- and beta 1-adrenergic receptors; however, similarities to the muscarinic cholinergic receptors are also evident. Two related genes have been identified by low stringency Southern blot analysis. These genes may represent additional alpha 2-adrenergic receptor subtypes.

Role of beta gamma subunits of G proteins in targeting the beta-adrenergic receptor kinase to membrane-bound receptors.

The rate and extent of the agonist-dependent phosphorylation of beta 2-adrenergic receptors and rhodopsin by beta-adrenergic receptor kinase (beta ARK) are markedly enhanced on addition of G protein beta gamma subunits. With a model peptide substrate it was demonstrated that direct activation of the kinase could not account for this effect. G protein beta gamma subunits were shown to interact directly with the COOH-terminal region of beta ARK, and formation of this beta ARK-beta gamma complex resulted in receptor-facilitated membrane localization of the enzyme. The beta gamma subunits of transducin were less effective at both enhancing the rate of receptor phosphorylation and binding to the COOH-terminus of beta ARK, suggesting that the enzyme preferentially binds specific beta gamma complexes. The beta gamma-mediated membrane localization of beta ARK serves to intimately link receptor activation to beta ARK-mediated desensitization.

Beta-arrestin-dependent formation of beta2 adrenergic receptor-Src protein kinase complexes.

The Ras-dependent activation of mitogen-activated protein (MAP) kinase pathways by many receptors coupled to heterotrimeric guanine nucleotide binding proteins (G proteins) requires the activation of Src family tyrosine kinases. Stimulation of beta2 adrenergic receptors resulted in the assembly of a protein complex containing activated c-Src and the receptor. Src recruitment was mediated by beta-arrestin, which functions as an adapter protein, binding both c-Src and the agonist-occupied receptor. beta-Arrestin 1 mutants, impaired either in c-Src binding or in the ability to target receptors to clathrin-coated pits, acted as dominant negative inhibitors of beta2 adrenergic receptor-mediated activation of the MAP kinases Erk1 and Erk2. These data suggest that beta-arrestin binding, which terminates receptor-G protein coupling, also initiates a second wave of signal transduction in which the "desensitized" receptor functions as a critical structural component of a mitogenic signaling complex.