Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1105 _____________________________ _____________________________ Alpha-2 Adrenergic Receptors and Signal Transduction Effector Output in Relation to G-Protein Coupling and Signalling Cross-Talk BY JOHNNY NÄSMAN ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2001 Dissertation for the Degree of Doctor of Philosophy (Faculty of Medicine) in Physiology presented at Uppsala University in 2002 ABSTRACT Näsman, J. 2001. Alpha-2 Adrenergic Receptors and Signal Transduction. Effector Output in Relation to G-Protein Coupling and Signalling Cross-Talk. Acta Universitatis Upsaliensis. Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1105. 64 pp. Uppsala. ISBN 91-554-5195-0. The alpha-2 adrenergic receptor (α2-AR) subfamily includes three different subtypes, α2A-, α2B- and α2C-AR, all believed to exert their function through heterotrimeric Gi/o-proteins. The present study was undertaken in order to investigate subtype differences in terms of cellular response and to explore other potential signalling pathways of α2-ARs. Evidence is provided for a strong Gs-protein coupling capability of the α2B-AR, leading to stimulation of adenylyl cyclase (AC). The difference between the α2A- and α2B-AR subtypes, in this respect, was shown to be due to differences in the second intracellular loops of the receptor proteins. Substitution of the second loop in α2A-AR with the corresponding domain of α2B-AR enrolled the chimeric α2A/α2B receptor with functional α2B-AR properties. Dual Gi and Gs coupling can have different consequences for AC output. Using coexpression of receptors and G-proteins, it was shown that the ultimate cellular response of α2B-AR activation is largely dependent on the ratio of Gi- to Gs-protein amounts in the cell. Also Gi- and Go-proteins appear to have different regulatory influences on AC. Heterologous expression of AC2 together with Gi or Go and the α2A-AR resulted in receptor-mediated inhibition of protein kinase C-stimulated AC2 activity through Go, whereas activation of Gi potentiated the activity. 2+ α2-ARs mobilize Ca in response to agonists in some cell types. This response was shown to depend on tonic purinergic receptor activity in transfected CHO cells. Elimination 2+ of the tonic receptor activity almost completely inhibited the Ca response of α2-ARs. In conclusion, α2-ARs can couple to multiple G-proteins, including Gi, Go and Gs. The cellular response to α2-AR activation depends on which receptor subtype is expressed, which cellular signalling constituents are engaged (G-proteins and effectors), and the signalling status of the effectors (dormant or primed). Key words: Adrenergic, receptor, G-protein, adenylyl cyclase, cAMP, calcium, cross-talk. Johnny Näsman, Department of Physiology, Uppsala University, Biomedical Center, P.O. Box 572, SE-751 23 Uppsala, Sweden Johnny Näsman 2001 ISSN 0282-7476 ISBN 91-554-5195-0 Printed in Sweden by Uppsala University, Tryck & Medier, Uppsala 2001 The thesis is based on the following papers, which are referred to in the text by their Roman numerals: I Dual signalling by different octopamine receptors converges on adenylate cyclase in Sf9 cells. Näsman J., Kukkonen J.P., Åkerman K.E.O. Insect Biochem. Mol. Biol., In press 2001. II The second intracellular loop of the α2-adrenergic receptors determines subtype-specific coupling to cAMP production. Näsman J., Jansson C., Åkerman K.E.O. J. Biol. Chem., 272, 9703-9708, 1997. III Role of G-protein availability in differential signaling by alpha 2- adrenoceptors. Näsman J., Kukkonen J.P., Ammoun S., Åkerman K.E.O. Biochem. Pharmacol., 62, 913-922, 2001. IV Endogenous extracellular purine nucleotides redirect α2-adrenoceptor signaling. Åkerman K.E.O., Näsman J., Lund P.-E., Shariatmadari R., Kukkonen J.P. FEBS Lett., 430, 209-212, 1998. V Modulation of adenylyl cyclase type 2 activity by Gi/o-protein coupled receptors. Opposite regulation by Gi and Go. Näsman J., Kukkonen J.P., Holmqvist T., Åkerman K.E.O. Manuscript 2001. Reprints were made with permission from the publishers. CONTENTS Abbreviations............................................................................................................ 3 1 Introduction ........................................................................................................... 4 1.1 General background............................................................................................ 4 1.2 G-protein coupled receptors and signal transduction......................................... 5 1.2.1 Receptors .............................................................................................. 6 1.2.2 Transducers .......................................................................................... 7 1.2.3 Effectors .............................................................................................10 1.2.3.1 Adenylyl cyclase, cAMP, and PKA.....................................10 1.2.3.2 Phospholipase C, Ca2+, and PKC .........................................13 1.3 α2-adrenergic receptors ....................................................................................15 1.3.1 A subfamily of adrenergic receptors ..................................................15 1.3.2 Physiological functions ......................................................................17 1.3.3 Cellular signalling ..............................................................................19 1.4 Other receptors utilized in the study.................................................................21 1.4.1 Octopamine receptors.........................................................................21 1.4.2 Purinergic receptors............................................................................22 1.4.3 Muscarinic receptors ..........................................................................23 1.5 Why study heterologously expressed receptors? .............................................23 2 Aims of the study.................................................................................................25 3 Materials and methods.........................................................................................26 3.1 Cell cultures...........................................................................................26 3.2 BEVS and infection procedures ............................................................26 3.3 Receptor quantification .........................................................................27 3.4 Receptor−G-protein interactions ...........................................................27 3.5 Measurement of cAMP .........................................................................28 3.6 Measurement of Ca2+.............................................................................29 3.7 Mathematical modelling........................................................................29 4 Results .................................................................................................................30 4.1 Receptor signalling in Sf9 cells.............................................................30 4.2 Propensity of α2-ARs for Gs coupling...................................................32 4.3 Signalling cross-talk..............................................................................34 5 Discussion............................................................................................................36 5.1 Suitability of Sf9 cells for receptor characterization.............................36 5.2 Promiscuous G-protein coupling...........................................................39 5.3 Adenylyl cyclase regulation by the Ca2+ signalling pathway ...............42 2+ 5.4 The α2-AR−Ca link ............................................................................43 6 Summary and conclusions...................................................................................46 7 Acknowledgements .............................................................................................48 8 References ...........................................................................................................50 ABBREVIATIONS AC adenylyl cyclase AR adrenergic receptor (as in α-AR and β-AR) BEVS Baculovirus Expression Vector System 2+ 2+ [Ca ]i intracellular concentration of free Ca CaM calmodulin CaMK calmodulin kinase CHO Chinese hamster ovary (a cell line) Ctx cholera toxin DAG diacylglycerol GPCR G-protein coupled receptor Gx-protein heterotrimeric GTP-binding protein with x identity of α subunit Gαx α subunit of heterotrimeric G-protein with x identity Gβγ βγ subunit complex of heterotrimeric G-protein i2/i3-loop second/third intracellular loop of receptors IBMX 3-isobutyl-1-methylxanthine IP3 inositol 1,4,5-trisphosphate PDE phosphodiesterase PH pleckstrin homology PI phosphtidylinositol PIP2 phosphtidylinositol 4,5-bisphosphate PK protein kinase (as in PKA and PKC) PL phospholipase (as in PLC, PLA2, PLD) Ptx pertussis toxin Sf9 Spodoptera frugiperda (a cell line) TM transmembrane domain of receptors TPA 12-O-tetradecanoyl phorbol-13-acetate 3 1 INTRODUCTION 1.1 General background All cells, whether single cell organisms or entities within multicellular organisms, need to sense the exterior to be able to adapt to changes. In the course of evolution, a multitude of such sensing molecules has evolved. A glimpse of this multitude comes from the realization that cells of our own bodies are regulated by a diversity of signals