The G-protein-coupled receptor kinases beta ARK1 and beta ARK2 are widely distributed at synapses in rat brain.

The beta-adrenergic receptor kinase (beta ARK) phosphorylates the agonist-occupied beta-adrenergic receptor to promote rapid receptor uncoupling from Gs, thereby attenuating adenylyl cyclase activity. Beta ARK-mediated receptor desensitization may reflect a general molecular mechanism operative on many G-protein-coupled receptor systems and, particularly, synaptic neurotransmitter receptors. Two distinct cDNAs encoding beta ARK isozymes were isolated from rat brain and sequenced. The regional and cellular distributions of these two gene products, termed beta ARK1 and beta ARK2, were determined in brain by in situ hybridization and by immunohistochemistry at the light and electron microscopic levels. The beta ARK isozymes were found to be expressed primarily in neurons distributed throughout the CNS. Ultrastructurally, beta ARK1 and beta ARK2 immunoreactivities were present both in association with postsynaptic densities and, presynaptically, with axon terminals. The beta ARK isozymes have a regional and subcellular distribution consistent with a general role in the desensitization of synaptic receptors.

An entirely cell-based system to generate single-chain antibodies against cell surface receptors.

The generation of recombinant antibodies (Abs) using phage display is a proven method to obtain a large variety of Abs that bind with high affinity to a given antigen. Traditionally, the generation of single-chain Abs depends on the use of recombinant proteins in several stages of the procedure. This can be a problem, especially in the case of cell-surface receptors, because Abs generated and selected against recombinant proteins may not bind the same protein expressed on a cell surface in its native form and because the expression of some receptors as recombinant proteins is problematic. To overcome these difficulties, we developed a strategy to generate single-chain Abs that does not require the use of recombinant protein at any stage of the procedure. In this strategy, stably transfected cells are used for the immunization of mice, measuring Ab responses to immunization, panning the phage library, high-throughput screening of arrayed phage clones, and characterization of recombinant single-chain variable regions. This strategy was used to generate a panel of single-chain Abs specific for the innate immunity receptor Toll-like receptor 2. Once generated, individual single-chain variable regions were subcloned into an expression vector allowing the production of recombinant Abs in insect cells, thus avoiding the contamination of recombinant Abs with microbial products. This cell-based system efficiently generates Abs that bind to native molecules on the cell surface, bypasses the requirement of recombinant protein production, and avoids risks of microbial component contamination.

The dopamine metabolite 3-methoxytyramine is a neuromodulator.

Dopamine (3-hydroxytyramine) is a well-known catecholamine neurotransmitter involved in multiple physiological functions including movement control. Here we report that the major extracellular metabolite of dopamine, 3-methoxytyramine (3-MT), can induce behavioral effects in a dopamine-independent manner and these effects are partially mediated by the trace amine associated receptor 1 (TAAR1). Unbiased in vivo screening of putative trace amine receptor ligands for potential effects on the movement control revealed that 3-MT infused in the brain is able to induce a complex set of abnormal involuntary movements in mice acutely depleted of dopamine. In normal mice, the central administration of 3-MT caused a temporary mild hyperactivity with a concomitant set of abnormal movements. Furthermore, 3-MT induced significant ERK and CREB phosphorylation in the mouse striatum, signaling events generally related to PKA-mediated cAMP accumulation. In mice lacking TAAR1, both behavioral and signaling effects of 3-MT were partially attenuated, consistent with the ability of 3-MT to activate TAAR1 receptors and cause cAMP accumulation as well as ERK and CREB phosphorylation in cellular assays. Thus, 3-MT is not just an inactive metabolite of DA, but a novel neuromodulator that in certain situations may be involved in movement control. Further characterization of the physiological functions mediated by 3-MT may advance understanding of the pathophysiology and pharmacology of brain disorders involving abnormal dopaminergic transmission, such as Parkinson's disease, dyskinesia and schizophrenia.

Mechanisms of specificity in neuronal activity-regulated gene transcription.

The brain is a highly adaptable organ that is capable of converting sensory information into changes in neuronal function. This plasticity allows behavior to be accommodated to the environment, providing an important evolutionary advantage. Neurons convert environmental stimuli into long-lasting changes in their physiology in part through the synaptic activity-regulated transcription of new gene products. Since the neurotransmitter-dependent regulation of Fos transcription was first discovered nearly 25 years ago, a wealth of studies have enriched our understanding of the molecular pathways that mediate activity-regulated changes in gene transcription. These findings show that a broad range of signaling pathways and transcriptional regulators can be engaged by neuronal activity to sculpt complex programs of stimulus-regulated gene transcription. However, the shear scope of the transcriptional pathways engaged by neuronal activity raises the question of how specificity in the nature of the transcriptional response is achieved in order to encode physiologically relevant responses to divergent stimuli. Here we summarize the general paradigms by which neuronal activity regulates transcription while focusing on the molecular mechanisms that confer differential stimulus-, cell-type-, and developmental-specificity upon activity-regulated programs of neuronal gene transcription. In addition, we preview some of the new technologies that will advance our future understanding of the mechanisms and consequences of activity-regulated gene transcription in the brain.

Functional evolution of mammalian odorant receptors.

The mammalian odorant receptor (OR) repertoire is an attractive model to study evolution, because ORs have been subjected to rapid evolution between species, presumably caused by changes of the olfactory system to adapt to the environment. However, functional assessment of ORs in related species remains largely untested. Here we investigated the functional properties of primate and rodent ORs to determine how well evolutionary distance predicts functional characteristics. Using human and mouse ORs with previously identified ligands, we cloned 18 OR orthologs from chimpanzee and rhesus macaque and 17 mouse-rat orthologous pairs that are broadly representative of the OR repertoire. We functionally characterized the in vitro responses of ORs to a wide panel of odors and found similar ligand selectivity but dramatic differences in response magnitude. 87% of human-primate orthologs and 94% of mouse-rat orthologs showed differences in receptor potency (EC50) and/or efficacy (dynamic range) to an individual ligand. Notably dN/dS ratio, an indication of selective pressure during evolution, does not predict functional similarities between orthologs. Additionally, we found that orthologs responded to a common ligand 82% of the time, while human OR paralogs of the same subfamily responded to the common ligand only 33% of the time. Our results suggest that, while OR orthologs tend to show conserved ligand selectivity, their potency and/or efficacy dynamically change during evolution, even in closely related species. These functional changes in orthologs provide a platform for examining how the evolution of ORs can meet species-specific demands.

When 7 transmembrane receptors are not G protein-coupled receptors.

Classically, 7 transmembrane receptors transduce extracellular signals by coupling to heterotrimeric G proteins, although recent in vitro studies have clearly demonstrated that they can also signal via G protein-independent mechanisms. However, the physiologic consequences of this unconventional signaling, particularly in vivo, have not been explored. In this issue of the JCI, Zhai et al. demonstrate in vivo effects of G protein-independent signaling by the angiotensin II type 1 receptor (AT1R) (see the related article beginning on page 3045). In studies of the mouse heart, they compare the physiologic and biochemical consequences of transgenic cardiac-specific overexpression of a mutant AT1R incapable of G protein coupling with those of a wild-type receptor. Their results not only provide the first glimpse of the physiologic effects of this newly appreciated mode of signaling but also provide important and previously unappreciated clues as to the underlying molecular mechanisms.

Identification of beta-adrenergic receptors in human lymphocytes by (-) (3H) alprenolol binding.

Human lymphocytes are known to posessess a catecholamine-responsive adenylate cyclase which has typical beta-adrenergic specificity. To identify directly and to quantitate these beta-adenergic receptors in human lymphocytes, (-) [3H] alprenolol, a potent beta-adrenergic antagonist, was used to label binding sites in homogenates of human mononuclear leukocytes. Binding of (-) [3H] alprenolol to these sites demonstrated the kinetics, affinity, and stereospecificity expected of binding to adenylate cyclase-coupled beta-adrenergic receptors. Binding was rapid (t1/2 less than 30 s) and rapidly reversible (t1/2 less than 3 min) at 37 degrees C. Binding was a saturable process with 75 +/- 12 fmol (-) [3H] alprenolol bound/mg protein (mean +/- SEM) at saturation, corresponding to about 2,000 sites/cell. Half-maximal saturation occurred at 10 nM (-) [3H] alprenolol, which provides an estimate of the dissociation constant of (-) [3H] alprenolol for the beta-adrenergic receptor. The beta-adrenergic antagonist, (-) propranolol, potently competed for the binding sites, causing half-maximal inhibition of binding at 9 nM. beta-Adrenergic agonists also competed for the binding sites. The order of potency was (-) isoproterenol greater than (-) epinephrine greater than (-)-norepinephrine which agreed with the order of potency of these agents in stimulating leukocyte adenylate cyclase. Dissociation constants computed from binding experiments were virtually identical to those obtained from adenylate cyclase activation studies. Marked stereospecificity was observed for both binding and activation of adenylate cyclase. (-)Stereoisomers of beta-adrenergic agonists and antagonists were 9- to 300-fold more potent than their corresponding (+) stereoisomers. Structurally related compounds devoid of beta-adrenergic activity such as dopamine, dihydroxymandelic acid, normetanephrine, pyrocatechol, and phentolamine did not effectively compete for the binding sites. (-) [3H] alprenolol binding to human mononuclear leukocyte preparations was almost entirely accounted for by binding to small lymphocytes, the predominant cell type in the preparations. No binding was detectable to human erythrocytes. These results demonstrate the feasibility of using direct binding methods to study beta-adrenergic receptors in a human tissue. They also provide an experimental approach to the study of states of altered sensitivity to catecholamines at the receptor level in man.

Trafficking of G protein-coupled receptors.

G protein-coupled receptors (GPCRs) play an integral role in the signal transduction of an enormous array of biological phenomena, thereby serving to modulate at a molecular level almost all components of human biology. This role is nowhere more evident than in cardiovascular biology, where GPCRs regulate such core measures of cardiovascular function as heart rate, contractility, and vascular tone. GPCR/ligand interaction initiates signal transduction cascades, and requires the presence of the receptor at the plasma membrane. Plasma membrane localization is in turn a function of the delivery of a receptor to and removal from the cell surface, a concept defined most broadly as receptor trafficking. This review illuminates our current view of GPCR trafficking, particularly within the cardiovascular system, as well as highlights the recent and provocative finding that components of the GPCR trafficking machinery can facilitate GPCR signaling independent of G protein activation.

Multiple endocytic pathways of G protein-coupled receptors delineated by GIT1 sensitivity.

Recently, we identified a GTPase-activating protein for the ADP ribosylation factor family of small GTP-binding proteins that we call GIT1. This protein initially was identified as an interacting partner for the G protein-coupled receptor kinases, and its overexpression was found to affect signaling and internalization of the prototypical beta(2)-adrenergic receptor. Here, we report that GIT1 overexpression regulates internalization of numerous, but not all, G protein-coupled receptors. The specificity of the GIT1 effect is not related to the type of G protein to which a receptor couples, but rather to the endocytic route it uses. GIT1 only affects the function of G protein-coupled receptors that are internalized through the clathrin-coated pit pathway in a beta-arrestin- and dynamin-sensitive manner. Furthermore, the GIT1 effect is not limited to G protein-coupled receptors because overexpression of this protein also affects internalization of the epidermal growth factor receptor. However, constitutive agonist-independent internalization is not regulated by GIT1, because transferrin uptake is not affected by GIT1 overexpression. Thus, GIT1 is a protein involved in regulating the function of signaling receptors internalized through the clathrin pathway and can be used as a diagnostic tool for defining the endocytic pathway of a receptor.