A. Donny Strosberg

Function and regulation of the β3-adrenoceptor

Abstract The cloning, sequencing and expression in model systems of the previously unidentified β 3 -adrenoceptor recently led to an extensive functional characterization. Ligand binding and adenylate cyclase activation studies helped define a specific profile that is quite distinct from that of the β 1 - and β 2 -adrenoceptors, but strongly reminiscent of most of the ‘atypical β-adrenoceptor-mediated responses reported in earlier pharmacological studies. More recently, a naturally occurring variation in the human β 3 -adrenoceptor has been correlated with hereditary obesity and with increased dynamic capacity to add on weight and develop non-insulin dependent diabetes in Western obese patients. Donny Strosberg and France Pietri-Rouxel describe how results now provide a consistent picture of an important role for the human β 3 -adrenoceptor in the regulation of lipid metabolism and as an obvious target for drugs to treat some forms of obesity and diabetes.

Differential signaling of human Mel1a and Mel1b melatonin receptors through the cyclic guanosine 3′-5′-monophosphate pathway

Cyclic guanosine 3-5-monophosphate (cGMP) has recently been shown to constitute a second messenger for Xenopus laevis melatonin Mel1c receptors. To verify whether cGMP levels are also modulated by mammalian melatonin receptors, we cloned the genes encoding the human Mel1a and Mel1b receptor subtypes and expressed them in human embryonic kidney cells. Pharmacological profiles and inhibition of forskolin-stimulated adenosine 3-5-cyclic monophosphate levels by melatonin confirmed functional expression of high-affinity melatonin receptors. Mel1b receptor-transfected cells modulated cGMP levels in a dose-dependent manner via the soluble guanylyl cyclase pathway. In contrast, Mel1a receptors had no effect on cGMP levels. These results demonstrate that mammalian melatonin receptors modulate cGMP levels and reveal for the first time differences in signaling between melatonin receptor subtypes, which may explain the necessity to express different receptor subtypes.

Growth Factor Activity of Endothelin-1 in Primary Astrocytes Mediated by Adhesion-Dependent and -Independent Pathways

Endothelin-1 (ET-1) has been shown to induce DNA synthesis in primary astrocytes by stimulating the extracellular signal-regulated kinase (ERK) pathway. To clarify the mechanisms responsible for the anchorage-dependent growth of astrocytes, the relationships between cell adhesion and ERK activation were investigated. Here it is reported that ET-1 promotes the formation of stress fibers and focal adhesions and the tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin, as well as Src activation and association of phosphorylated FAK with Grb2. Pretreatment of astrocytes with cytochalasin D or C3-transferase, which inhibits actin polymerization or Rho activity, respectively, prevented the activation/phosphorylation of Src, FAK, and paxillin after ET-1 stimulation; by contrast, the ERK pathway was not significantly affected. This differential activation of FAK/Src and ERK pathways was also observed with astrocytes 10 and 60 min after replating on poly-l-ornithine-precoated dishes. Collectively, these findings indicate that activation of FAK and Src is dependent on actin cytoskeleton integrity, Rho activation, and adhesion to extracellular matrix, whereas ERK activation is independent of these intracellular events and seems to correlate with activation of the newly identified protein tyrosine kinase PYK2. Induction of DNA synthesis by ET-1, however, was reduced dramatically in astrocytes pretreated with either cytochalasin D or C3-transferase. This study provides a demonstration of Rho- and adhesion-dependent activation of FAK/Src, which collaborates with adhesion-independent activation of PYK2/ERK for DNA synthesis in ET-1-stimulated astrocytes.

In vitro degradation of the C-terminal octapeptide of cholecystokinin by ‘enkephalinase A’

As the C‐terminal octapeptide of cholecystokinin represents a putative neurotransmitter in the central nervous system, the membrane‐bound enzymes involved in its inactivation were investigated. Two aminopeptidases, involved in the cleavage of enkephalins, and a metalloendopeptidase were identified in extracts of solubilized synaptic membranes. The metalloendopeptidase, which cleaves CCK‐8 at the Trp30—Met31 bond, appeared to be indistinguishable from ‘enkephalinase A1’ on the basis of its chromatographic behaviour, sensitivity to inhibitors and relative affinities for Met‐ and Leu‐enkephalins. This finding indicates that CCK‐8 is inactivated in vitro by the same peptidases as enkephalins.

Comparison of serological tests for the diagnosis of feline immunodeficiency virus infection of cats

A new enzyme-linked-immunosorbent assay (ELISA) for the detection of antibodies to feline immunodeficiency virus was compared with previously described ELISAs. Serum samples from 184 infected or uninfected cats were tested using a whole virus lysate kit and ELISAs based on recognition of one of two synthetic peptides (P237 and P253) localized in the transmembrane domain of the viral envelope. The whole virus lysate commercial kit led to the detection of 6% false positive and 4.3% false negative sera. The ELISA based on peptide P253 gave no false positive result and failed to detect only one serum that was subsequently shown to be positive by radio-immunoprecipitation assay. A sandwich-ELISA test using Galanthus nivalis agglutinin, a lectin that specifically binds terminal mannose groups of the envelope proteins was used as a confirmatory test for equivocal results with peptide ELISA and gave similar results. This study indicates that recognition of P253 could serve as a sensitive and specific test for the diagnosis of seropositivity to feline immunodeficiency virus, and moreover that the Galanthus nivalis ELISA could be useful in equivocal cases as a confirmatory test.

Immortalized Rat Brain Microvessel Endothelial Cells: II-Pharmacological Characterization

Vascular endothelial cells are known to display a variety of biological functions, including regulation of vascular tone through secretion of vasoactive factors and control of nutrient and cellular trafficking between the blood and the underlying tissue (1,2). Most of the available data concerning these functions have been collected in vitro from studies on macrovascular peripheral endothelial cells. Given the remarkable heterogeneity of endothelia in terms of morphology and function, we intended to assess the ability of brain microvessel endothelial cells, which constitute the blood-brain barrier (BBB), to secrete endothelin (ET-1) and nitric oxide (NO), two potent vasoactive factors, under the control of hormonal stimuli and to express the MHC molecules that may contribute to the adhesion of immune cells to the endothelium. This study was performed with immortalized rat brain microvessel endothelial cells that were isolated after transfection of primary cultures with the E1A-Adenovirus encoding gene. These cells present a non-transformed phenotype and express the blood-brain barrier markers γ-glutamyl transferase and alkaline phosphatase during angiogenesis, as described in the accompanying paper (Roux et al.).

The iodocyanopindolol and SM-11044 binding protein belongs to the TM9SF multispanning membrane protein superfamily.

SM-11044 is the only beta-adrenergic agonist that inhibits guinea pig eosinophil chemotaxis and induces relaxation of depolarized rat colon tonus. We have previously reported the purification of a 34 kDa photoaffinity-labeled SM-11044 binding protein (SMBP) from rat colon that may mediate the biological effects of the ligand and that differs from all known monoamine receptors (Sugasawa et al., J. Biol. Chem. 272 (1997) 21244). The present report describes partial amino acid sequence of rat SMBP and molecular cloning of corresponding human SMBP (hSMBP) cDNA. This cDNA encodes a 588 amino acid residue polypeptide comprising a signal peptide, a long hydrophilic amino-terminal region, and a highly hydrophobic C-terminal portion organized into nine putative transmembrane domains. The sequence and structure of hSMBP shows homology to members of a new transmembrane protein 9 superfamily (TM9SF). Comparison of hSMBP with related protein sequences from yeast, plant and human revealed two subgroups within TM9SF. The members of these groups differ in length and have characteristic amino acid sequence motifs in their amino-terminal portion. Northern blot analysis revealed two major SMBP mRNAs, at 3.4 and 3.8 kb, that were present in all the human tissues examined. Western blot experiments detected SMBP as a 70 kDa protein that may be further cleaved into an active 34 kDa N-terminal polypeptide. Stable Chinese Hamster Ovary cell transfectants expressing hSMBP cDNA displayed specific binding of [(125)I]iodocyanopindolol that was displaced by SM-11044 in a dose-dependent manner. Thus, SMBP is the first member of TM9SF with functional ligand binding properties, suggesting that some of these integral membrane proteins may function as channels, small molecule transporters or receptors.

The HIV-1 Nef Protein Inhibits Extracellular Signal-Regulated Kinase-Dependent DNA Synthesis in a Human Astrocytic Cell Line

Abstract: The role of nonproductive infection of astrocytes by human immunodeficiency virus type 1 (HIV‐1), characterized by the overexpression of nef, in brain disease progression is largely unknown. We investigated the consequences of stable expression of nef from the HIV‐1 strain LAI in the human astrocytic cell line U373. DNA synthesis induced by endothelin‐1 (ET‐1) was largely decreased by nef. Stable expression of nef did not affect the ET‐1‐induced tyrosine phosphorylation of focal adhesion kinase, an adhesion‐dependent pathway known to participate in DNA synthesis in astrocytes. Conversely, the activation of extracellular signal‐regulated kinase (ERK) by ET‐1 was largely inhibited in cells stably or transiently expressing nef. A similar inhibitory action of nef on ERK activation was observed after direct stimulation of G proteins. Furthermore, the inhibitory action of nef did not require protein kinase C (PKC) and affected mainly the PKC‐independent pathway of ERK activation. Following chemokine receptor CXCR4‐mediated infection of U373 cells stably expressing CXCR4 with the T‐tropic HIV‐1 strain m7‐NDK, ET‐1‐induced activation of ERK was also inhibited. Altogether, these results indicate that intracellular signaling pathways associated with the growth factor activity of ET‐1 are impaired in nef‐expressing and HIV‐1‐infected astrocytes, suggesting that infection of astrocytes may play a significant role in the neuropathogenesis of HIV‐1 encephalopathy.

Genomic cloning and species-specific properties of the recombinant canine β3-adrenoceptor

A molecular clone encoding a beta3-adrenoceptor was isolated from a canine genomic library. The cloned receptor exhibited a pharmacological profile similar to that of other species: in particular, high efficiency of the two selective beta3-adrenoceptor agonists, CL 316,243 (disodium(R,R)-5[2[[2-(chlorophenyl)-2hydroxyethyl]-amino]propyl]- 1,3-benzodioxole-2,2-dicarboxylate) and ICI 201651 ((R)4-(2-hydroxy-3-phenoxypropylaminoethoxy)-N-(2-methoxyethyl)phe noxy acetic acid) and a low affinity for the radioligand (-)-[3-(125)I]-iodocyanopindolol. Interestingly, CGP 12177A ((+/-)-4-(3-t-butylamino-2-hydroxypropoxy)benzimidazol-2-one), which is described as a partial agonist for the human receptor, was a full agonist for the canine receptor. After expression and stimulation of the canine beta3-adrenoceptor in stably transfected Chinese hamster ovary cells there was a very low accumulation of cAMP, suggesting weak coupling to Gs-protein and adenylyl cyclase. However, the response was much better in human embryonal kidney cells transfected with the canine beta3-adrenoceptor gene. The cloning of the canine beta3-adrenoceptor and the insights gained from its pharmacological characterization may allow the development of selective compounds for use in the treatment of obese dogs.

High Level Functional Expression of Human β1-Adrenergic Receptor in Baculovirus-Infected Cells Screened by a Rapid in Situ Procedure

A novel screening assay for the identification of baculovirus infected cells expressing membrane receptors was developed by using a replica transfer technique. Sf9 cells were cotransfected with wild type baculoviral DNA and the transfer vector pVL941-beta 1 containing the coding region of the human beta 1-adrenergic receptor gene. Infected cells embedded in agarose were incubated with [125I]-iodocyanopindolol and transferred onto filters that were subsequently autoradiographed. This procedure resulted in the isolation of recombinant baculoviruses that expressed beta 1-adrenergic receptors. Binding assays carried out with [125I]-ICYP indicated that more than 600,000 receptors were expressed per cell, the highest level noted so far for this receptor in genetically engineered cells. Sf9 cells expressing the beta 1-AR were analysed by ligand binding, competition experiments, adenylyl cyclase stimulation and photoaffinity labeling. These cells express a homogenous population of receptors and display the known pharmacological properties of beta 1-AR in human tissues.