3-3 -q1 IuBxrrFrcATroN' CUIRACTERISATION' AND MorEcuLAR ANlLYsrs oF THE ATTnRNATIvELY SPLrcEn FONUS OF THE LUTBINIZING HONMONB RNCEPTOR rN THB OvrNE OvIRY DBIN J. BnclcH B.Sc. (HoNs) A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy Department of Obstetrics and Gynaecology, The FacultY of Medicine. UniversitY of Adelaide, Adelaide, South Australia J , 1996 Enur¿, At the time of submission of this thesis, the paper "Mutational analyses of the extracellular domain of the full-length Lutropin/Choriogonadotropin receptor suggests leucine-rích repeats 1-6 are involved in hormone binding" by T.homas, D., Rozell, T.G., Liu, X. and Segaloff, D.L. (1996) in Molecular Endocrinology 10:76O-768, was unavailable to the author. This paper further defines the region of LTVCG receptor that is involved in high affimty binding to hCG andLH. i.e. from amino acids -26 to 206 (Braun et al., 1991; see Tøble 1.7) to -26 to 164, with amino acids 165 to2ll being mildly important. Ío Denise, tuty Çuiling Lígfit in tfr¿, DørLest 9{ours ff not; T^lnln or ConTENTS Table of Contents................ .l Summary.. .xiii Declaration .xvi Acknowledgments .xvii Publications................ .xix Conference Presentations ............. .xx Awards .xxii List of Abbreviations used in this Thesis' .xxiii CH¡prnn ONr: Rrvrnw or rHE LnnR-lrunn 2 1. I Introduction.......... J 1.2 Physiology of the Ovary........ 1.2.1 From Primordial Germ cells to Primordial Follicles J 1.2.2 F ollicular DeveloPment 4 1.2.3 Ovulation ......... 1.2.4 Resumption of Meiosis 1.2.5 4tresia.............. 1.2.6 Follicular Steroidogenesis and its Control by Cell Type 1.2.7 The CorPus Luteum 1.2.8 Luteal Regression..........'..... 7.2.9 Lttteal Function during Pregnancy 1 . 3 Luteinizing Hormone and Chorionic Gonadotrophin......'.... " " " " 1.3.1 Regulaton of LH secretion'.' I . 3.2 Regulation of Chorionic Gonadotrophin secretion" "" " I.4LWCG Receptors. 1.4.1 Location of the LWCG Receptor... ............24 1.4.1. 1 The Testes ...,.',..'..24 1.4.1.2 Non-Gonadal Tissue ............24 l.4.2Primary Structure and Topology of the LIVCG Receptor ............28 1.4.3 Cloning of the LIVCG Receptor .,,.........33 1.4.4 Structural Organisation of the LI{/CG Receptor Gene..'..........".. ............37 1.4.5 Homology of the LIVCG Receptor with the FSH and TSH Receptors 1.4.6 Functional Properties of the LIVCG Receptor... 1.4.6.1 G Protein Activation 1.4.6.2 The cAMP PathwaY 1.4.6.3 The Phospholipase C Pathway 1.4.7 Regulation of the Functional Properties and Numbers of LIICG Receptors. 51 1.4.7.1. Uncoupling of LFVCG receptors... 52 Heterologous Uncoupling of the LIVCG receptor'........ 52 Homologous Uncoupling of the LTVCG receptor.......... s2 1 .4.7 .2. Up-Regulation of LIVCG Receptors................ 55 1,4.7 .3. Down-Regulation of the LH Receptor ......'...'.'. 57 Down-Regulation of LH Receptors in Follicles 57 Down-Regulation of LH Receptors in Luteal cells...'... 59 Down-Regulation in Leydig Cells and Leydig Tumor CeIls......... 6l 1.4.8 Structural Functions of the LIICG Receptor 65 A. Hormone binding 66 1. Amino Acid Determinants....,... 66 2. Carbohydrate Determinants 73 B. Effector Activation 76 1l 1.4.9. Heterogeneity of LFVCG Receptor Messenger RNA Transcripts ....79 1.5 Alternative Splicing............... ....81 1.5. 1. Mechanisms of Alternative Splicing'..'.........-. ....81 1.5.2. Alternative Splicing of the LFVCG Receptor Gene..' ....82 1,6 Hypothesis and Aims of the Thesis ....89 Cuerrnn Two: GnNnnel Mnrnnws AND Mtrnons 2.0 Reagents and Solutions.............. 92 2.1 Solutions for Molecular BiologY 92 2.1.1 Molecular Biology Stock Reagents and Buffers..'.'....'.'.... 92 Alkaline Lysis Buffers for Mini Preparations of Plasmid DNA......... 92 5.7 MCaesium Chloride (CsCl) Solution 93 10x Calf Intestinal Alkaline Phosphatase buffer 93 Deionised Formamide 93 50x Denhardt's solution .......'....... 93 Diethylpyrocarbonate (DEPC) treated II2O ......... 94 I M Dithiothreitol (DTT) 94 6x DNA loading buffer...'..' 94 40 mM dNTP solution ,94 Ethidium Bromide ,94 0.5 M Ethylenediaminetetra Acetic Acid (EDTA), pH 8.0..." .95 3.1 M GTC solution............. .9s Isopropylthio-B-D-galactoside (IPTG).. .. .. .95 X-gal .95 Ligase 10x buffer .96 ru Maxiprep Lytic Mix. ..96 Northern Blot Prehybridisation mixture ..96 Northern Blot Stripping Solution.............',. ..96 Phenol ..96 Phenol : Chloroform : Isoamyl alcohol...'.. ..97 Proteinase K Buffer ..97 Qiagen Buffers for Large Scale Plasmid Preparations .'.,... ..97 RNase (for general use) ..97 lOx RNA loading buffer ..98 RNase Protection Hybridisation buffer....... .. ..' ...98 RNase Protection Digestion mixture ...98 RNase Protection Formamide Loading buffer ...98 10x RNA Transblot Buffer ...98 RNA Methylene Blue stain...'...... ...99 20x SSC ...99 20x SSPE ...99 STE Buffer................ ...99 I 0x Tris-Acetate-EDTA (TAE) Buffer........... _..99 I 0x Tris-Borate-EDTA (TBE) Buffer'.. "'.. -.'.' ....99 2.l.2Bacterial Media ,...100 Ampicillin ,...100 I M CaCl2 Stocks ....100 20o/o Casamino acids. ....100 1 M MgCl2 Stocks ....100 I M MgSO4 Stocks ....100 10x M9 Salt Solution............... ....100 Glucose/lvlinimal Medium Agar Plates ......'.. ....101 Luria Bertani (LB) Medium...'..'..... ..:.101 lv Superbroth Medium.... .. r01 Special Broth Medium.... ..101 SOB Medium ............,. ..101 SOG Medium............ ..t02 TFB buffer ..102 Preparation of LB plates with ampicillin........... ..102 Converting LB Amp plates into X-gal plates........ ..102 2.2 Molecular Biology Techniques................ ..103 2.2.1 Plasmid Purification .........,.. ..103 2.2.1.1 Small Scale Plasmid Preparation . Alkaline Lysis......,.. .. 103 2.2. 1 .2 Large Scale Plasmid Preparations................ ..104 Large Scale Plasmid Preparation and Purification by CsCl gradient centrifugation........ ..104 Maxi Preparation of Plasmid DNA : Qiagen Method.... ..105 2.2.2 Restriction Endonuclease Di gestion. ., ., ., .'.. ..105 2.2.3 General procedures for RNase-free conditions. ..106 DEPC-treatment of soluttons... ...106 2.2.4 Preparation of Total RNA......... ...106 RNA Isolation by CsCl Gradient Centrifugation............ ...107 RNA Isolation by the Morrison Method'.... ...107 2.2.5 Quantitation of DNA and RNA ...108 2.2.6 Polymerase Chain Reaction (PCR)....... ..,109 Primer Design.... ...109 Synthesis and isolation of oligonucleotides. ...109 Standard amplífi cation conditions ...111 2.2.7 Fiev erse-Transcriptase Polymerase Chain Reaction (RT-PCR) ...111 2.2.8 Gel Electrophoresis.............., ...1t2 Agarose gel electroPhoresis ...t12 v Polyacrylamide gel electrophoresis ..........' tt2 2.2.9 Synthesis of RNA probes....... tt2 In vitro transcription of RNA probes....... t12 Determining 32p-Utp incorporation ............. ll3 2.2.10 RNA Analysis by Northern Blotting.... 113 Preparation of samPles 113 RNA electrophoresis for Northern blotting tt4 Electrotransfer of RNA to nylon filters tt4 Staining nylon filters for RNA.... l14 Filter hybridisation.............. 115 Washing the filter and autoradiography.'.. 11s Re-use of blots 1r6 2.2.1I Cloning and subcloning of DNA fragments........'....... ,116 Isolation of DNA fragments from TAE gels and from solution 116 End filling restriction enzyme generated 5' overhangs .. "..'..'...... l17 Dephosphorylation of linearised vector DNA 118 Lígation of DNA 118 2.2.12 Transformation of E. Coli cells .......... 118 Hanahan's Transformation of E col¡ ljll4l09 118 Preparation of competent DH5cr, E. coli cells for electroporation............ 119 Transformation of competent DH5a E- Col¡ cells by electroPoration............ 119 2.2.I3 Analysis of Transformants...... r20 Screening bacterial colonies for recombinant plasmids using PCR.......... ..120 2.2.14 Sequencing ..t21 VI Sequencing gels ........... t2r 2.2.15 RNase Protection assays 12l Analysis of RNase Protection Assays 123 2.3 Solutions for Polyclonal Antibody Production t23 2.3.1 Polyclonal Antibody Production Stock Solutions. 123 Phosphate Buffered Saline (PBS) 123 Normal Saline 123 Antibody AssaY Buffer....'.. 123 2.4 Polyclonal Antibody Production Techniques ......... t23 2.4.1 Peptide design. 123 2.4.2 Animals............. 124 2.4.3 Immunisation Protocol.... t24 2.4.4 Collection of Serum........... t2s 2.4.5 lodination of peptides t25 IodogenrM coating of tubes .t25 Iodination of peptides .t2s Purification of the iodinated peptides.,. .126 2.4.6 Isolation of IgG .126 2.4.7 Determining Antibody Titre ......... .127 2.5 Protein Analysis Solutions.. .127 2.5.1 Protein Analysis Stock Solutions.. .127 Transblot buffer .t27 Acrylamide gel stain .127 Acrylamide gel destain .............. .128 Acrylamide gel Preservative .128 SDS-Acrylamide Gel Running Buffer .. .t28 2x Denaturing Sample Buffer............... .128 4x SDS Running Gel Buffer .t28 vll 4x SDS Stacking Gel Buffer Tris-Tricine SDS-PAGE Anode buffer.............. Tris-Tricine SDS-PAGE Cathode buffer ...... "'.. 3x Tris-Tricine SDS-PAGE Gel buffer..... Nitrocellulose Protein Stain.... "... Nitrocellulose Protein Destain....' Homogenisation Buffer 4".......... Homogenisation Buffer B ..'......... Homogenisation Buffer C.........'.. Western Blot Buffers'.'.'..."'..' Bouins Fixative Solution.... 2.6 Protein Analysis... 2.ó. I Protein Isolation General Homogenisation Method..'........' Microsomal Preparation ....'.'........ 2.6.2 Quantitation of Proteins 2.6.3 lodination of Goat anti-Rabbit Antibodies 2.6.4 SDs-Polyacrylamide Gel Electrophoresis . Staining Acrylamide Gels 2.6.5 Protein Analysis