ß-Adrenergic receptor structure and function: molecular insights guiding the development of novel therapeutic strategies to treat malignancy.

ß adrenergic receptors mediate effects via activation of G proteins, transactivation of membrane growth factor receptors, or ß adrenergic receptor-ß arrestin-facilitated scaffold-mediated signaling. Agonist occupancy of the ß adrenergic receptor induces desensitization by promoting ß adrenergic receptor kinase phosphorylation of the carboxyl terminal domain, facilitating binding of the amino terminal of the ß arrestin, which sterically inhibits interactions between ß adrenergic receptors and G proteins and induces clathrin-coated pit-mediated receptor endocytosis. Scaffold formation promoted by ß arrestin binding to the ß adrenergic receptor activates extracellular regulated kinase 1/2 in a manner which elicits cytosolic retention of, and prevents promotion of nuclear transcriptional activity by, mitogen-activated protein kinase. The ß adrenergic receptor kinase also interacts with a yet to be determined microsomal membrane protein via high-affinity electrostatic interactions. We evaluate ß adrenergic receptor structure, function, and downstream signaling and ß arrestin-mediated desensitization, receptor endocytosis, and scaffold-facilitated signal transduction in order to illumine therapeutic strategies designed to modulate these pathways. We trust these approaches may arm us with the capacity to selectively modulate signal transduction pathways regulating cellular proliferation, immunogenicity, angiogenesis, and invasive and metastatic potential implicated in cancer initiation, promotion, and progression.

Ultrastructural relations between beta-adrenergic receptors and catecholaminergic neurons.

We performed dual electron microscopic immunocytochemistry to determine the precise cellular relations between beta-adrenergic receptors (beta AR) and catecholaminergic terminals within adult rat brains. An antibody, beta AR404, against a peptide corresponding to the C-terminus of the hamster lung beta AR (beta 2 subtype) together with an anti-tyrosine hydroxylase (TH), a catecholaminergic marker, were used. Results show predominant labeling for beta AR404 within small astrocytic processes (beta-A). This is in sharp contrast to earlier results which showed neuronal labeling when using antibodies against the third intracellular loop of the receptor and of neurons-plus-astrocytes labeled using antibodies against the whole beta AR molecule. beta-A within visual cortex and nuclei of the solitary tracts frequently contacted blood vessel basement membrane and TH-immunoreactive terminals. TH-immunoreactive axons forming axo-axonic juxtapositions with non-TH terminals were also noted to be surrounded by beta-A. In the area postrema, a brain region lacking a blood-brain barrier, few beta-A occurred adjacent to TH-immunoreactive terminals or elsewhere. Thus, 1) catecholamines may act beyond morphologically identifiable synapses; 2) beta-A may mediate interactions between catecholamines and other transmitters; 3) there may be substantial heterogeneity in the structure or the conformation of the beta AR protein between neurons and glia or across CNS regions.

Three-dimensional structure for the beta 2 adrenergic receptor protein based on computer modeling studies.

Computer-aided model building techniques have been used to construct three-dimensional model structures for hamster beta 2 adrenergic receptor. Experimental data were used as constraints to guide the model building procedure, and a number of rather strict criteria were applied to assess the physical plausibility of model structures. We present details of our best model structure to date, which is consistent with a large body of experimental data. We also discuss in detail our model building procedures and evaluation criteria, which we believe may be of general utility in modeling projects.

Morphology and distribution of neurons and glial cells expressing beta-adrenergic receptors in developing kitten visual cortex.

The morphology and distribution of cells expressing beta-adrenergic receptors has been studied in developing kitten visual cortex using a monoclonal antibody which recognizes both beta-1 and beta-2 adrenergic receptors. We found specific populations of neurons and glial cells which express beta-adrenergic receptor immunoreactivity in the kitten visual cortex. In adult animals, the receptors are most concentrated in the superficial and deep cortical layers (layers I, II, III and VI). About 50% of the stained neural cells in adult cat visual cortex are glial cells. Most of the immunoreactive neurons in layers III and V are pyramidal cells while those in layers II and IV are more likely to be nonpyramidal cells. In neonatal kittens, staining is weaker than that in adult cats and it appears to be concentrated in neurons of the deep cortical layers and in the subcortical plate and white matter. Only a few immunoreactive glial cells were found at this age. Receptor numbers increase after birth and by 24 days of age, the laminar distribution of beta-adrenergic receptors approaches that of adult animals. Immunoreactive glial cells in the white matter show a progressive increase in number throughout postnatal development.

Detrimental effects of beta-adrenergic stimulation on beta-adrenoceptors and microtubules in the heart.

Increased plasma catecholamines - in particular, excessive beta-adrenoceptor activation in chronic heart failure - may easily desensitize the beta-adrenoceptors as well as the postreceptor signal transductions. Since these detrimental changes in the failing heart could be reversible, administration of low-dose beta-blocker, which minimizes the negative inotropic effects, may be effective in attenuating the harmful effects of sympathetic nerve activation. Beta-adrenoceptor stimulation may also produce microtubule disruptions of the cell either through direct action or through an increase in heart rate. Treatment with beta-blockers could attenuate Ca overload by slowing the heart rate and may be useful as a protection from the structural disintegration of the cell. Thus, to clarify the underlying mechanisms of beta-blocker therapy for chronic heart failure, we have to consider not only to the functional aspects but also to the structural changes of the cells.

Altered patterns of agonist-stimulated cAMP accumulation in cells expressing mutant beta 2-adrenergic receptors lacking phosphorylation sites.

As with many other receptor-effector systems, the responsiveness of the beta-adrenergic receptor (beta AR)/adenylyl cyclase system undergoes desensitization upon agonist exposure. Phosphorylations of the receptor by the cAMP-dependent protein kinase (protein kinase A) and the beta AR kinase appear to play roles in such desensitization phenomena, but the functional significance of the receptor phosphorylation in intact cells has not been previously assessed. In this study, we constructed and expressed in a mammalian fibroblast line the normal (wild type) human beta 2 AR and mutant forms of the receptor that lack the putative phosphorylation sites for these two protein kinases. The two consensus sequences for phosphorylation by protein kinase A were altered by changing serines 261, 262 and 345, 346 to alanines. In another mutant, the 11 serines and threonines at the carboxy terminus of the protein that constitute the putative beta AR kinase phosphorylation sites were changed to alanines or glycines. The mutated receptors did not differ from the wild type in their affinities for agonists or antagonists or in their ability to mediate agonist stimulation of adenylyl cyclase. Moreover, their levels of expression in the cultured cells were the same. When stimulated with the beta AR agonist isoproterenol, cells bearing either the wild type or mutant receptors generated cAMP at essentially identical rates for the first 2 min. Cells bearing wild type receptors then showed a rapid desensitization characterized by a markedly diminished rate of cAMP production after the first few minutes of stimulation. However, cells bearing either of the mutated forms of the receptor showed much less desensitization and continued to generate cAMP at a rate 3-4 times greater than that observed in cells expressing the wild type receptor. In contrast, intact cell cAMP levels stimulated by prostaglandin E1 and forskolin were not different between cells bearing wild type or mutant beta AR. These results suggest an important physiological role for phosphorylation of the beta AR in regulating rapid agonist-induced desensitization in intact cells.

Identification of a specific site required for rapid heterologous desensitization of the beta-adrenergic receptor by cAMP-dependent protein kinase.

The molecular basis for heterologous desensitization of the beta-adrenergic receptor (beta AR) was investigated by site-directed mutagenesis of the beta AR protein. Rapid heterologous desensitization of agonist-stimulated adenylyl cyclase activity was observed when L cells expressing the wild-type beta AR were incubated with 50 nM epinephrine. This desensitization response could be mimicked in a cell-free system by incubation with cAMP-dependent protein kinase (cA.PK). Deletion of amino acid residues 259-262 from the beta AR, removing one of the two consensus sequences in the receptor for phosphorylation by cA.PK, abolished the ability of the receptor to undergo rapid heterologous desensitization. In contrast, deletion of the other cA.PK consensus sequence (residues 343-348) or truncation of the Ser/Thr-rich C-terminal tail of the beta AR (deletion of residues 354-418) did not affect this heterologous desensitization process. These results suggest that the action of cA.PK on amino acid residue(s) contained within the sequence 259-262 of the beta AR is required for rapid heterologous desensitization of the receptor in response to agonists.

Impaired desensitization of cardiac beta-adrenoceptors in the spontaneously hypertensive rat.

Reduced inotropic responsiveness by spontaneously hypertensive rats (SHR) to beta agonists is associated with, and may be secondary to, a decline in cardiac membrane-bound beta adrenoceptors. It is widely assumed that this decline reflects a desensitization of the receptor secondary to enhanced sympathetic drive, but direct evidence is lacking. We examined this issue in cell-free cardiac preparations from age-matched SHR and Wistar-Kyoto (WKY) rats. Isoproterenol induced a time- and concentration-dependent decline in membrane-bound beta receptors but was considerably more effective in WKY than in SHR (43% decline versus 29% in SHR, P less than 0.01). Since cyclic AMP-protein kinase inhibitor prevented only 15-20% of the isoproterenol-induced decline, this was probably mediated by the beta receptor kinase. Cyclic AMP-dependent protein kinase induced a 38% desensitization in WKY rats but was completely ineffective in SHR. These results suggest that desensitization of the beta receptor is impaired in SHR, probably because the structural organization of the receptor in the cardiac membranes is altered.

Autoradiographic localization of beta-adrenergic receptors in rat oviduct.

Catecholamines are known to have an inhibitory effect on oviductal smooth musculature by changing the adrenergic receptors activity. In order to further investigate the role of epinephrine and/or norepinephrine in oviduct, the distribution of beta-adrenergic receptors has been studied in the rat oviduct, using in vitro autoradiography and [125I]cyanopindolol (CYP), as radioligand. The specificity of the labelling and the characterization of receptor subtypes in different cell types was achieved by displacement of radioligand with increasing concentrations of zinterol, a beta-adrenergic agonist with preferential affinity for the beta 2-adrenoreceptor subtype and practolol, a beta-adrenergic antagonist that binds preferentially to beta 1-subtype. Quantitative estimation of ligand binding was achieved by densitometry. It was shown that the vast majority of beta-receptors were of the beta 2-subtype and were found in smooth muscle layers as well as in the epithelium. The latter localization suggests a role for epinephrine and/or norepinephrine on the oviductal epithelium.

Effects of unilateral decortication on beta-adrenergic receptors in the remaining cortex and in the hypothalamus of female rats.

Many experiments have been performed to evaluate the physiological role of catecholaminergic mechanisms in gonadotropin release. The purpose of the present study was to determine the concentration of beta-adrenoreceptors in the remaining (right) cerebral cortex and in right and left hypothalamic halves of hemi-decorticated female rats which exhibited elevated plasma gonadotropin levels as observed previously. The density of beta-receptors was measured using a high-affinity beta-adrenergic ligand, iodocyanopindolol (ICYP). Scatchard estimates were obtained for maximum binding (Bmax fmol/mg of tissues) from pooled cerebral cortical and hypothalamic tissue of animals under several experimental conditions after hemi-decortication and sham operation. There was an increase in beta-adrenoreceptor density in the remaining (right) cerebral cortex at all times examined in hemi-decorticate in comparison with the sham-operated animals (7 days, +10.9%; 21 days, +8.4%; 90 days, +22%; and 90 days plus ovariectomy, +34.8%). The number of beta-adrenoreceptors in the right hypothalamic half in hemi-decorticates decreased at 21 days (-42.20%) and then increased at 90 days (+76.63%) and 90 days plus ovariectomy (+51.75%) when compared with the left hypothalamic half. At the same time there were no significant changes in the sham-operated animals when comparing the receptor density in the right and left hypothalamic halves, respectively. Thus, our results suggest a direct or indirect adrenergic pathway by which the left cortex can influence the right cortex and a crossed pathway to the contralateral hypothalamus changing adrenergic activity which can alter the beta-adrenergic receptor binding capacity in the hypothalamus.

Autoradiographic visualization of beta-adrenoceptor subtypes in human lung.

Adrenoceptor subtypes have been localized in human lung by an autoradiographic method, using [125I]iodocyanopindolol (ICYP) to label beta-receptors in tissue sections. The ICYP was incubated with cryostat sections of microscopically normal human lung method on microscope slides for 2 h at 37 degrees C. Nonspecific binding was determined by incubating adjacent serial sections in the presence of 200 microM (-)-isoproterenol. Specific binding, which accounted for greater than 90% of total counts bound to the sections, was saturable, of high affinity, and stereoselective, having the same pharmacologic characteristics as binding to homogenates prepared from the same lungs. Competition with selective beta-receptor antagonists ICI 118,551 (beta 2-selective) and betaxolol (beta 1-selective) showed that the ratio of beta 2 to beta 1-receptors in the sections was approximately 3:1. Autoradiography revealed that beta-receptors were widely distributed, with dense labeling over airway epithelium, alveolar walls, and submucosal glands, and a lower density of grains over airway and vascular smooth muscle. The beta-receptors of airway smooth muscle from large and small airways were entirely of the beta 2-receptor subtype, which is consistent with functional studies. Similarly, beta-receptors of airway epithelium and vascular smooth muscle were also entirely of the beta 2-receptor subtype. In bronchial submucosal glands and alveolar walls, both receptor subtypes appeared to coexist with beta 2-receptors, making up 10% of the total in glands and 30% in alveoli. The significance of beta 1- and beta 2-receptors in alveolar walls (which account for greater than 90% of total beta-receptors in human lung) remains to be determined.

[Molecular physiology of adrenoreceptors]. / K molekuliarnoi fiziologii adrenoretseptorov.

The paper reviews some problems of theoretical investigation of the spatial of adrenergic drugs and their receptors, citing results recently obtained by the authors. It includes the chapters as follows: Introduction; Conformation - activity relationships of ligands; Theoretical conformational analysis; Noradrenaline; Classification and some models of adrenergic receptors; Problems of ligands productive conformation selection and the description of receptor topography; Principles of choice of ligands; Criteria for productive conformation selection; Productive conformations of beta 2-adrenergic agonists and beta 2-receptor topography; Productive conformations of alpha 1-adrenergic receptor ligands and alpha 1-receptor topography; Structure-activity relationships of some ligands in the light of adrenergic beta 2- and alpha 1-receptors topography; Possible role of calcium ion in the interaction of adrenergic agonists with receptors.