Calmodulin-binding sites on adenylyl cyclase type VIII

Ca2+ stimulation of adenylyl cyclase type VIII (ACVIII) occurs through loosely bound calmodulin. However, where calmodulin binds in ACVIII and how the binding activates this cyclase have not yet been investigated. We have located two putative calmodulin-binding sites in ACVIII. One site is located at the N terminus as revealed by overlay assays; the other is located at the C terminus, as indicated by mutagenesis studies. Both of these calmodulin-binding sites were confirmed by synthetic peptide studies. The N-terminal site has the typical motif of a Ca2+-dependent calmodulin-binding domain, which is defined by a characteristic pattern of hydrophobic amino acids, basic and aromatic amino acids, and a tendency to form amphipathic alpha-helix structures. Functional, mutagenesis studies suggest that this binding makes a minor contribution to the Ca2+ stimulation of ACVIII activity, although it might be involved in calmodulin trapping by ACVIII. The primary structure of the C-terminal site resembles another calmodulin-binding motif, the so-called IQ motif, which is commonly Ca2+-independent. Mutagenesis and functional assays indicate that this latter site is a calcium-dependent calmodulin-binding site, which is largely responsible for the Ca2+ stimulation of ACVIII. Removal of this latter calmodulin-binding region from ACVIII results in a hyperactivated enzyme state and a loss of Ca2+ sensitivity. Thus, Ca2+/calmodulin regulation of ACVIII may be through a disinhibitory mechanism, as is the case for a number of other targets of Ca2+/calmodulin.     

RGS2 interacts with Gs and adenylyl cyclase in living cells

Regulator of G Protein Signalling (RGS) proteins impede heterotrimeric G protein signalling. RGS2 decreases cAMP production and appears to interact with both adenylyl cyclase (AC) and its stimulatory G protein Gs. We showed previously that Green Fluorescent Protein-tagged RGS2 (GFP-RGS2) localizes to the nucleus in HEK 293 cells and is recruited to the plasma membrane when co-expressed with Gsalpha, or the Gs-coupled beta2-adrenergic receptor (beta2AR). Here, using confocal microscopy we show that co-expression of various AC isoforms (ACI, ACII, ACV, ACVI) also leads to GFP-RGS2 recruitment to the plasma membrane. Bioluminescence Resonance Energy Transfer (BRET) was also used to examine physical interactions between RGS2 and components of the Gs-signalling pathway. A BRET signal was detected between fusion constructs of RGS2-Renilla luciferase (energy donor) and Gsalpha-GFP (energy acceptor) co-expressed in HEK 293 cells. BRET was also observed between GFP-RGS2 and ACII or ACVI fused to Renilla luciferase. Additionally, RGS2 was found to interact with the beta2AR. Purified RGS2 selectively bound to the third intracellular loop of the beta2AR in GST pulldown assays, and a BRET signal was observed between GFP-RGS2 and beta2AR fused to Renilla luciferase when these two proteins were co-expressed together with either ACIV or ACVI. This interaction was below the limit of detection in the absence of co-expressed AC, suggesting that the effector enzyme stabilized or promoted binding between the receptor and the RGS protein inside the cell. Taken together, these results suggest the possibility that RGS2 might bind to a receptor-G protein-effector signalling complex to regulate Gs-dependent cAMP production.     

An isoform-specific interaction of the membrane anchors affects mammalian adenylyl cyclase type V activity.

The nine membrane-bound mammalian adenylyl cyclases (ACs) contain two highly diverged membrane anchors, M1 and M2, with six transmembrane spans each and two conserved cytosolic domains which coalesce into a pseudoheterodimeric catalytic unit. Previously, the catalytic segments, bacterially expressed as soluble proteins, were characterized extensively whereas the function of the membrane domains remained unexplored. Using the catalytic C1 and C2 domains of AC type V we employed the membrane anchors from type V and VII ACs for construction of enzymes with duplicated, inverted, fully swapped and chimeric membrane anchors. Further, in the M1 membrane domain individual transmembrane spans were removed or exchanged between type V and VII ACs. The constructs were expressed in HEK293 cells, the expression levels and membrane localization was assessed by Western blotting. Cell-free basal, forskolin-, GTP gamma S-and G(s alpha)/GTP gamma S-stimulated AC activities were determined. The results demonstrate that enzymatic activities were only maintained when the M1 and M2 membrane domains were derived from either AC V or VII. Constructs with chimeric membrane domains, i.e. M1 from type V and M2 from type VII AC or vice versa, were essentially inactive although the expression levels and membrane localization appeared to be normal. The data indicate a functionally important interaction of the membrane domains of ACs in that they seem to interact in a pair-like, isoform delimited manner. This interaction directly impinges on the formation of the catalytic interface. We propose that protein-protein interactions of the AC membrane domains may constitute another, yet unexplored level of AC regulation.     

Mechanism of Galpha i-mediated inhibition of type V adenylyl cyclase

The topology of mammalian adenylyl cyclase reveals an integral membrane protein composed of an alternating series of membrane and cytoplasmic domains (C1 and C2). The stimulatory G protein, Galpha(s), binds within a cleft in the C2 domain of adenylyl cyclase while Galpha(i) binds within the opposite cleft in the C1 domain. The mechanism of these two regulators also appears to be in opposition. Activation of adenylyl cyclase by Galpha(s) or forskolin results in a 100-fold increase in the apparent affinity of the two domains for one another. We show herein that Galpha(i) reduces C1/C2 domain interaction and thus formation of the adenylyl cyclase catalytic site. Mutants that increase the affinity of C1 for C2 decrease the ability of Galpha(i) to inhibit the enzyme. In addition, Galpha(i) can influence binding of molecules to the catalytic site, which resides at the C1/C2 interface. Adenylyl cyclase can bind substrate analogs in the presence of Galpha(i) but cannot simultaneously bind Galpha(i) and transition state analogs such as 2'd3'-AMP. Galpha(i) also cannot inhibit the membrane-bound enzyme in the presence of manganese, which increases the affinity of adenylyl cyclase for ATP and substrate analogs. Thus homologous G protein alpha-subunits promote bidirectional regulation at the domain interface of the pseudosymmetrical adenylyl cyclase enzyme.     

Chromosomal mapping of human adenylyl cyclase genes type III,type V and type VI

Adenylyl cyclase activity plays a central role in the regulation of most cellular processes. At least eight different adenylyl cyclases have been identified, which are endowed with various and sometimes opposing regulatory properties. Recently we have localized the human genes encoding two of these adenylyl cyclases: the gene for type 11 adenylyl cyclase is located on chromosome 2 (sub-band 2p15.3), the gene for type VIII is located on chromosome 8 (sub-band 8824.2). More recently the type I gene has been located on chromosome 7 (sub-band 7pl2–7p13). Using in situ hybridization, we have now localized the genes for three other adenylyl cyclases: the type III gene has been localized on chromosome 2 in the sub-band 2p22–2p24, the type V gene on chromosome 3 at position 3q13.2–3q21, and the type VI gene on chromosome 12 at position 12q12–12q13. It therefore appears that all adenylyl cyclase genes, known at present are located on different chromosomes and thus are likely to be independently regulated.     

Two interaction sites on mammalian adenylyl cyclase type I and II: modulation by calmodulin and G(betagamma)

Mammalian ACs (adenylyl cyclases) are integrating effector molecules in signal transduction regulated by a plethora of molecules in either an additive, synergistic or antagonistic manner. Out of nine different isoforms, each AC subtype uses an individual set of regulators. In the present study, we have used chimaeric constructs, point mutations and peptide competition studies with ACs to show a common mechanism of multiple contact sites for the regulatory molecules G(betagamma) and calmodulin. Despite their chemical, structural and functional variety and different target motifs on AC, G(betagamma) and calmodulin share a two-site-interaction mechanism with G(alphas) and forskolin to modulate AC activity. Forskolin and G(alphas) are known to interact with both cytosolic domains of AC, from inside the catalytic cleft as well as at the periphery. An individual interaction site located at C(1) of the specifically regulated AC subtype had been ascribed for both G(betagamma) and calmodulin. In the present study we now show for these two regulators of AC that a second isoform- and regulator-specific contact site in C(2) is necessary to render enzyme activity susceptible to G(betagamma) or calmodulin modulation. In addition to the PFAHL motif in C(1b) of ACII, G(betagamma) contacts the KF loop in C(2), whereas calmodulin requires not only the Ca2+-independent AC28 region in C(1b) but also a Ca2+-dependent domain in C(2a) of ACI containing the VLG loop to stimulate this AC isoform.     

Cell-specific properties of type V and type IX adenylyl cyclase isozymes in 293T cells and embryonic chick ventricular myocytes

The cDNAs for types V and IX adenylyl cyclases were cloned from a chicken heart library and expressed in 293T cells (plasmid transfection) and in embryonic chick ventricular myocytes (adenovirus infection). Expression of type V or IX cyclases in 293T cells resulted in increases in basal and isoproterenol (ISO)-stimulated cAMP levels, whereas the expression of type V, but not type IX, cyclase increased forskolin (FK)-stimulated cAMP levels. Expression of type V cyclase in cardiac myocytes increased basal and FK-stimulated cAMP levels, variably increased ISO-stimulated cAMP levels, and decreased the content of beta-adrenergic receptors (betaARs). The expression of type IX cyclase in cardiac myocytes increased basal and ISO-elevated cAMP levels and, surprisingly, increased the cAMP-elevating effect of FK. The finding that FK responses are increased in cardiac myocytes but not in 293T cells expressing the type IX cyclase suggests that the host cell influences the properties of the type IX isozyme.     

Identification of regulatory residues of the yeast adenylyl cyclase.

We have attempted to identify amino acid residues of the yeast adenylyl cyclase that are involved in the regulation of its activity, by isolating adenylyl cyclase-linked spontaneous mutations capable of suppressing the temperature-sensitive phenotype of ras1- ras2-ts1 strains. We previously identified a mutated adenylyl cyclase in which a single point mutation, called CR14, led to the replacement of threonine 1651 with isoleucine. We have now investigated the biological effects of CR14, and of other mutations that cause the replacement of threonine 1651 by distinct amino acids. We have observed that the response of adenylyl cyclase to Ras can be either enhanced or attenuated, without significant effects on the steady-state level of the former enzyme in vivo, depending on the amino acid side chain at position 1651. Therefore, this residue identifies a regulatory region on the adenylyl cyclase molecule. We have also taken advantage of the attenuation of adenylyl cyclase function caused by the replacement of threonine 1651 with aspartic acid to isolate intragenic suppressor mutations. We have identified several point mutations, leading to single amino acid substitutions, individually capable of reactivating the attenuated adenylyl cyclase. The corresponding amino acid changes are located within a relatively small region, including residues 1331, 1345, 1348 and 1374. This region could be physiologically involved in the negative control of the carboxy-terminal catalytic domain.     

Modulation of adenylyl cyclase activity in young and adult rat brain cortex. Identification of suramin as a direct inhibitor of adenylyl cyclase

Adenylyl cyclase (AC) in brain cortex from young (12-day-old) rats exhibits markedly higher activity than in adult (90-day-old) animals. In order to find some possibly different regulatory features of AC in these two age groups, here we modulated AC activity by dithiothreitol (DTT), Fe(2+), ascorbic acid and suramin. We did not detect any substantial difference between the effects of all these tested agents on AC activity in cerebrocortical membranes from young and adult rats, and the enzyme activity was always about two-fold higher in the former preparations. Nevertheless, several interesting findings have come out of these investigations. Whereas forskolin- and Mn(2+)-stimulated AC activity was significantly enhanced by the addition of DTT, increased concentrations of Fe(2+) ions or ascorbic acid substantially suppressed the enzyme activity. Lipid peroxidation induced by suitable combinations of DTT/Fe(2+) or by ascorbic acid did not influence AC activity. We have also observed that PKC- or protein tyrosine kinase-mediated phosphorylation apparently does not play any significant role in different activity of AC determined in cerebrocortical preparations from young and adult rats. Our experiments analysing the presumed modulatory role of suramin revealed that this pharmacologically important drug may act as a direct inhibitor of AC. The enzyme activity was diminished to the same extent by suramin in membranes from both tested age groups. Our present data show that AC is regulated similarly in brain cortex from both young and adult rats, but its overall activity is much lower in adulthood.     

Olfactory adenylyl cyclase. Identification and purification of a novel enzyme form

Rat olfactory adenylyl cyclase has been identified by means of a monoclonal antibody BBC-2, which reacts with both Ca2+/calmodulin-sensitive and -insensitive forms of adenylyl cyclase (Mollner, S., and Pfeuffer, T. (1988) Eur. J. Biochem. 171, 265-271). The antibody recognized a 180-kDa polypeptide in olfactory cilia but not in decilitated olfactory epithelial membranes. A protein of the same mobility was observed when olfactory adenylyl cyclase was purified by forskolin-agarose affinity chromatography followed by radioiodination. Its identity was further established by cross-linking to [32P]ADP-ribosylated Gs alpha (GTP-binding protein), to yield a single radiolabeled product of Mr approximately 220. Olfactory adenylyl cyclase has a approximately 3-fold higher turnover number, as assessed from stoichiometric binding of [35S]guanosine 5'-(3-O-thio)triphosphate. Therefore, the considerably higher specific adenylyl cyclase activity in olfactory cilia must be due to a approximately 100-fold higher molar concentration of enzyme in this tissue.     

Modeling of Galpha(s) and Galpha(i) regulation of human type V and VI adenylyl cyclase

We examined the kinetics of Galpha(s) and Galpha(i) regulation of human type V and type VI adenylyl cyclase (AC V and AC VI) in order to better model interactions between AC and its regulators. Activation of AC VI by Galpha(s) displayed classical Michaelis-Menten kinetics, whereas AC V activation by Galpha(s) was cooperative with a Hill coefficient of 1.4. The basal activity of human AC V, but not that of AC VI, was inhibited by Galpha(i). Both enzymes showed greater inhibition by Galpha(i) at low Galpha(s) concentrations; however, human AC V was activated by Galpha(i) at high Galpha(s) concentrations. Neither regulator had an effect on the K(m) for Mg-ATP. Mutations made within the Galpha(s) binding pocket of AC V (N1090D) and VI (F1078S) displayed 6- and 14-fold greater EC(50) values for Galpha(s) activation but had no effect on Galpha(i) inhibition of basal activity or K(m) for Mg-ATP. Galpha(s)-stimulated AC VI-F1078S was not significantly inhibited by Galpha(i), despite normal inhibition by Galpha(i) upon forskolin stimulation. Mechanistic models for Galpha(s) and Galpha(i) regulation of AC V and VI were derived to describe these results. Our models are consistent with previous studies, predicting a decrease in affinity of Galpha(i) in the presence of Galpha(s). For AC VI, Galpha(s) is required for inhibition but not binding by Galpha(i). For AC V, binding of two molecules of Galpha(s) and Galpha(i) to an AC dimer are required to fully describe the data. These models highlight the differences between AC V and VI and the complex interactions with two important regulators.     

Gbetagamma activation site in adenylyl cyclase type II. Adenylyl cyclase type III is inhibited by Gbetagamma

The Gbetagamma complex of heterotrimeric G proteins is the most outstanding example for the divergent regulation of mammalian adenylyl cyclases. The heterodimeric Gbetagamma complex inhibits some isoforms, e.g. ACI, and stimulates the isoforms ACII, -IV, and -VII. Although former studies identified the QEHA region located in the C2 domain of ACII as an important interaction site for Gbetagamma, the determinant of the stimulatory effect of Gbetagamma has not been detected. Here, we identified the C1b domain as the stimulatory region using full-length adenylyl cyclase. The relevant Gbetagamma signal transfer motif in IIC1b was determined as MTRYLESWGAAKPFAHL (amino acids 493-509). Amino acids of this PFAHL motif were absolutely necessary for ACII to be stimulated by Gbetagamma, whereas they were dispensable for Galpha(s) or forskolin stimulation. The PFAHL motif is present in all three adenylyl cyclase isoforms that are activated by Gbetagamma but is absent in other adenylyl cyclase isoforms as well as other known effectors of Gbetagamma. The emerging concept of two contact sites on different molecule halves for effective regulation of adenylyl cyclase is discussed.     

The role of calmodulin recruitment in Ca2+ stimulation of adenylyl cyclase type 8

Ca2+ stimulation of adenylyl cyclase type 8 (AC8) is mediated by calmodulin (CaM). An earlier study identified two CaM binding sites in AC8; one that was apparently not essential for AC8 activity, located at the N terminus, and a second site that was critical for Ca2+ stimulation, found at the C terminus (Gu, C., and Cooper, D. M. F. (1999) J. Biol. Chem. 274, 8012-8021). This study explores the role of these two CaM binding domains and their interaction in regulating AC8 activity, employing binding and functional studies with mutant CaM and modified AC8 species. We report that the N-terminal CaM binding domain of AC8 has a role in recruiting CaM and that this recruitment is essential to permit stimulation by Ca2+ in vivo. Using Ca2+-insensitive mutants of CaM, we found that partially liganded CaM can bind to AC8, but only fully liganded Ca2+/CaM can stimulate AC8 activity. Moreover, partially liganded CaM inhibited AC8 activity in vivo. The results indicate that CaM pre-associates with the N terminus of AC8, and we suggest that this recruited CaM is used by the C terminus of AC8 to mediate Ca2+ stimulation.     

Type-specific regulation of adenylyl cyclase. Selective pharmacological stimulation and inhibition of adenylyl cyclase isoforms

Crystallographic studies have elucidated the binding mechanism of forskolin and P-site inhibitors to adenylyl cyclase. Accordingly, computer-assisted drug design has enabled us to identify isoform-selective regulators of adenylyl cyclase. After examining more than 200 newly synthesized derivatives of forskolin, we found that the modification at the positions of C6 and C7, in general, enhances isoform selectivity. The 6-(3-dimethylaminopropionyl) modification led to an enhanced selectivity for type V, whereas 6-[N-(2-isothiocyanatoethyl) aminocarbonyl] and 6-(4-acrylbutyryl) modification led to an enhanced selectivity for type II. In contrast, 2'-deoxyadenosine 3'-monophosphate, a classical and 3'-phosphate-substituted P-site inhibitor, demonstrated a 27-fold selectivity for inhibiting type V relative to type II, whereas 9-(tetrahydro-2-furyl) adenine, a ribose-substituted P-site ligand, showed a markedly increased, 130-fold selectivity for inhibiting type V. Consequently, on the basis of the pharmacophore analysis of 9-(tetrahydro-2-furyl) adenine and adenylyl cyclase, a novel non-nucleoside inhibitor, 2-amino-7-(2-furanyl)-7,8-dihydro-5(6H)-quinazolinone (NKY80), was identified after virtual screening of more than 850,000 compounds. NKY80 demonstrated a 210-fold selectivity for inhibiting type V relative to type II. More importantly, the combination of a type III-selective forskolin derivative and 9-(tetrahydro-2-furyl) adenine or NKY80 demonstrated a further enhanced selectivity for type III stimulation over other isoforms. Our data suggest the feasibility of adenylyl cyclase isoform-targeted regulation of cyclic AMP signaling by pharmacological reagents, either alone or in combination.     

Mapping of adenylyl cyclase genes type I,II, III,IV, V,and VI in mouse

The adenylyl cyclases (AC) act as second messengers in regulatory processes in the central nervous system. They might be involved in the pathophysiology of diseases, but their biological function is unknown, except for AC type I, which has been implicated in learning and memory. We previously mapped the gene encoding AC I to human Chromosome (Chr) 7p12. In this study we report the mapping of the adenylyl cyclase genes type I–VI to mouse chromosomes by fluorescence in situ hybridization (FISH): Adcy1 to Chr 11A2, Adcy2 to 13C1, Adcy3 to 12A-B, Adcy4 to 14D3, Adcy5 to 16B5, and Adcy6 to 15F. We also confirmed previously reported mapping results of the corresponding human loci ADCY2, ADCY3, ADCY5, and ADCY6 to human chromosomes and, in addition, determined the chromosomal location of ADCY4 to human Chr 14q11.2. The mapping data confirm known areas of conservation between mouse and human chromosomes.     

Quantitative reduction of type I adenylyl cyclase in human alcoholics.

The amounts of adenylyl cyclase type I (AC I) were examined in various parts of the postmortem brains from alcoholics who prior to death had been abstinent from alcohol for at least 6 months and compared with controls using immunoblot analysis with anti-AC I specific antibody. It was revealed that a significant reduction of AC I was observed in both frontal and temporal cortices. On the other hand, in other areas (occipital cortex, caudate nucleus, putamen, and hippocampus) the amounts were comparable between alcoholics and controls. In the next step, we examined two subtypes of human AC mRNA levels (AC I and AC VIII) in blood cells by quantitative RT-PCR using [alpha-32P]dCTP with two sets of the synthetic oligonucleotide primers based on the DNA sequences reported elsewhere (Villacres, E.C. et al., Genomics 16 (1993) 473-478; J. Parma et al., Biochem. Biophys. Res. Commun. 179 (1991) 455-462). The amounts of amplified DNAs of both AC I and AC VIII were significantly smaller in alcoholics than in controls. On the other hand, the amounts of amplified DNA of beta-actin DNA were almost equal between alcoholics and controls. It appears from these results that a reduction in the amount of AC subtypes may be a biological marker for alcoholics.     

Effect of association with adenylyl cyclase-associated protein on the interaction of yeast adenylyl cyclase with Ras protein.

Posttranslational modification of Ras protein has been shown to be critical for interaction with its effector molecules, including Saccharomyces cerevisiae adenylyl cyclase. However, the mechanism of its action was unknown. In this study, we used a reconstituted system with purified adenylyl cyclase and Ras proteins carrying various degrees of the modification to show that the posttranslational modification, especially the farnesylation step, is responsible for 5- to 10-fold increase in Ras-dependent activation of adenylyl cyclase activity even though it has no significant effect on their binding affinity. The stimulatory effect of farnesylation is found to depend on the association of adenylyl cyclase with 70-kDa adenylyl cyclase-associated protein (CAP), which was known to be required for proper in vivo response of adenylyl cyclase to Ras protein, by comparing the levels of Ras-dependent activation of purified adenylyl cyclase with and without bound CAP. The region of CAP required for this effect is mapped to its N-terminal segment of 168 amino acid residues, which coincides with the region required for the in vivo effect. Furthermore, the stimulatory effect is successfully reconstituted by in vitro association of CAP with the purified adenylyl cyclase molecule lacking the bound CAP. These results indicate that the association of adenylyl cyclase with CAP is responsible for the stimulatory effect of posttranslational modification of Ras on its activity and that this may be the mechanism underlying its requirement for the proper in vivo cyclic AMP response.     

Conformation-dependent activation of type II adenylyl cyclase by protein kinase C

Phorbol ester treatment enhanced the catalytic activity of type II adenylyl cyclase overexpressed in insect cells. In cells coexpressing type II adenylyl cyclase and protein kinase C-α, type II adenylyl cyclase catalytic activity was higher even in the absence of phorbol ester treatment; phorbol ester treatment further and markedly enhanced type II adenylyl cyclase catalytic activity. However, this enhancement, either by phorbol ester treatment or by coexpression of protein kinase C-α, was lost following membrane solubilization with detergents. This attenuation was unaffected by phosphatase inhibitor or salts. In contrast, membrane solubilization did not affect forskolin-stimulated type II adenylyl cyclase catalytic activity. Purified type II adenylyl cyclase was stimulated by forskolin and Gsα, but not by protein kinase C-α. Therefore, a specific mammalian protein kinase C isoenzyme can activate type II adenylyl cyclase, but the mechanism clearly differs from that underlying either Gsα- or forskolin-mediated stimulation. J. Cell. Biochem. 64:492–498. © 1997 Wiley-Liss, Inc.     

Phenolamine-dependent adenylyl cyclase activation in Drosophila Schneider 2 cells

Drosophila Schneider 2 (S2) cells are often employed as host cells for non-lytic, stable expression and functional characterization of mammalian and insect G-protein-coupled receptors (GPCRs), such as biogenic amine receptors. In order to avoid cross-reactions, it is extremely important to know which endogenous receptors are already present in the non-transfected S2 cells. Therefore, we analyzed cellular levels of cyclic AMP and Ca2+, important second messengers for intracellular signal transduction via GPCRs, in response to a variety of naturally occurring biogenic amines, such as octopamine, tyramine, serotonin, histamine, dopamine and melatonin. None of these amines (up to 0.1 mM) was able to reduce forskolin-stimulated cyclic AMP production in S2 cells. Furthermore, no agonist-induced calcium responses were observed. Nevertheless, the phenolamines octopamine (OA) and tyramine (TA) induced a dose-dependent increase of cyclic adenosine monophosphate (AMP) production in S2 cells, while serotonin, histamine, dopamine and melatonin (up to 0.1 mM) did not. The pharmacology of this response was similar to that of the octopamine-2 (OA2) receptor type. In addition, this paper provides evidence for the presence of an endogenous mRNA encoding an octopamine receptor type in these cells, which is identical or very similar to OAMB. This receptor was previously shown to be positively coupled to adenylyl cyclase.