Mechanisms of
Signal
and Cell
,
,H1: INTRODUCTION: CORE PRINCIPLES ............................................................................................................................2
1.1 GENERAL INTRODUCTION .................................................................................................................................................... 2
1.2 THE MAIN PRINCIPLES OF CELL SIGNALING ............................................................................................................................... 2
1.2.1 The main idea ....................................................................................................................................................... 2
1.2.2 Many extra components ....................................................................................................................................... 3
1.2.3 Signal transduction has different outcomes ......................................................................................................... 3
1.2.4 Points to keep in mind........................................................................................................................................... 3
1.3 CHANGE IS THE KEY ............................................................................................................................................................ 5
1.4 WHAT MAKES A GOOD SIGNAL? ............................................................................................................................................ 5
1.5 DIFFERENT WAYS IN WHICH CELLS SIGNAL TO EACH OTHER .......................................................................................................... 6
1.5.1 Electrical ............................................................................................................................................................... 6
1.5.2 Chemical ............................................................................................................................................................... 6
1.6 MECHANISMS OF CELL-TO-CELL SIGNALING ............................................................................................................................. 8
1.6.1 Direct cell-cell signaling ........................................................................................................................................ 8
1.6.2 Receptor-ligand signaling – cell-surface receptor ................................................................................................. 8
1.6.3 Vesicle-mediated signaling ................................................................................................................................. 11
1.7 SPATIAL AND TEMPORAL CONTROL IN CELL SIGNALING ............................................................................................................. 12
1.7.1 Interorganelle communication ........................................................................................................................... 12
1.7.2 Protein sorting and translocation ....................................................................................................................... 13
1.7.3 Protein modifications and organelle environments ............................................................................................ 14
1.7.4 Protein degradation ............................................................................................................................................ 15
1.7.5 Temporal and spatial control in cell signaling .................................................................................................... 15
1.7.6 Modular nature of signaling pathways .............................................................................................................. 15
1.8 DOMAINS AND MODULES .................................................................................................................................................. 16
1.9 CELLULAR SIGNALING IS NOT ONLY A MATTER OF CLASSICAL PROTEINS ......................................................................................... 17
1.9.1 The concept of signal amplification .................................................................................................................... 17
1.9.2 Feedback control and crosstalk .......................................................................................................................... 17
1.9.3 Non-proteins in cell signaling transduction ........................................................................................................ 17
H2: FIRST MESSENGERS ................................................................................................................................................. 18
2.1 SIGNALS AND RECEPTORS .................................................................................................................................................. 18
2.1.1 Terminology ........................................................................................................................................................ 18
2.1.2 Types of signals ................................................................................................................................................... 18
2.1.3 Eukaryotic organelles and cellular structures involved in cell signaling ............................................................. 18
2.2 CELL-TO-CELL COMMUNICATION ......................................................................................................................................... 19
2.3 SYNAPTIC TRANSMISSION .................................................................................................................................................. 20
2.3.1 The synapse ........................................................................................................................................................ 20
2.3.2 Neurotransmitters .............................................................................................................................................. 21
2.4 HORMONES .................................................................................................................................................................... 24
2.4.1 Specific hormones and their biological effects.................................................................................................... 24
2.4.2 Mode of action ................................................................................................................................................... 25
2.4.3 Hormonal regulation in insects ........................................................................................................................... 27
2.4.4 Plant hormones................................................................................................................................................... 28
H3: RECEPTORS .............................................................................................................................................................. 29
3.1 GENERAL INTRODUCTION AND PROPERTIES OF RECEPTORS ....................................................................................................... 29
3.2 LIGAND-RECEPTOR BINDING ............................................................................................................................................... 29
3.3 RECEPTOR SENSITIVITY AND RECEPTOR DENSITY...................................................................................................................... 31
3.3.1 Receptor sensitivity ............................................................................................................................................. 31
3.3.2 Receptor density ................................................................................................................................................. 32
3.4 RECEPTOR BINDING ASSAYS ................................................................................................................................................ 32
3.4.1 Steps in a receptor binding assay ....................................................................................................................... 33
3.4.2 Separation of bound and free ligand? ................................................................................................................ 33
3.5 DIFFERENT KINDS OF RECEPTORS, LIGANDS AND RESPONSES ..................................................................................................... 34
3.5.1 Different kinds of receptors ................................................................................................................................ 34
, 3.5.2 Different kinds of ligands .................................................................................................................................... 34
3.5.3 Different kinds of responses................................................................................................................................ 35
3.5.4 Molecular mechanisms behind signal transduction ........................................................................................... 35
3.6 INTRACELLULAR RECEPTORS ............................................................................................................................................... 36
3.6.1 Nuclear receptor family ...................................................................................................................................... 36
3.7 MEMBRANE RECEPTORS .................................................................................................................................................... 37
3.7.1 Ligand-gated ion channels .................................................................................................................................. 37
3.7.2 Single transmembrane receptors ........................................................................................................................ 38
3.7.3 Heptahelical protein receptors: G-protein coupled receptors (GPCRs) ............................................................... 39
3.8 LIGAND-RECEPTOR ASSAYS ................................................................................................................................................. 42
A. Scintillation proximity assay (SPA) ........................................................................................................................... 42
B. Cytosensor microphysiometer ................................................................................................................................. 42
C. Genetically engineered cell line ............................................................................................................................... 42
D. Intracellular calcium measurements with fluorescent dyes .................................................................................... 42
E. Co-expression with promiscuous Gα subunits ......................................................................................................... 42
F. Fluorescent imaging plate reader (FLIPR) ................................................................................................................ 42
G. Aequorin-based functional screening ...................................................................................................................... 42
H. Genetically encoded calcium or cAMP sensors ....................................................................................................... 42
I. cAMP sensor using YFP-Epac-RLuc (CAMYL) ............................................................................................................. 42
J. Bioluminescence resonance energy transfer (BRET) ................................................................................................. 42
K. NanoBiT protein-protein interaction system ............................................................................................................ 42
H4: PROTEIN PHOSPHORYLATION, KINASES AND PHOSPHATASES .................................................................................. 43
4.1 PHOSPHORYLATION .......................................................................................................................................................... 43
4.1.1 Covalent modification regulates enzyme activity ............................................................................................... 43
4.1.2 Phosphorylation? ................................................................................................................................................ 43
4.2 PROTEIN KINASES............................................................................................................................................................. 45
4.2.1 Serine/Threonine kinases.................................................................................................................................... 47
4.2.2 Tyrosine kinases .................................................................................................................................................. 55
4.3 PHOSPHATASES ............................................................................................................................................................... 57
H5: MODULATORS OF G-PROTEIN SIGNALING, CYCLASES AND PHOSPHODIESTERASES .................................................. 58
5.1 INTRODUCTION ............................................................................................................................................................... 58
5.1.1 GPCR and G-protein structure ............................................................................................................................ 58
5.1.2 Signaling and G protein subunits ........................................................................................................................ 58
5.2 REGULATORS OF GPCRS – RECEPTOR-LEVEL MODULATION....................................................................................................... 59
5.2.1 RAMPs – Receptor-Activity Modifying Proteins .................................................................................................. 59
5.2.2 GPCR desensitization .......................................................................................................................................... 59
5.2.3 G-protein receptor kinases (GRKs) ...................................................................................................................... 59
5.2.4 α- and β-arrestins ............................................................................................................................................... 60
5.3 REGULATORS OF G-PROTEINS – SUBUNIT-LEVEL MODULATION .................................................................................................. 61
5.3.1 Regulators of G-protein signaling (RGS) ............................................................................................................. 61
5.3.2 GoLoco motifs ..................................................................................................................................................... 61
5.3.3 Non-GPCR mediated activation of Gα proteins by GIV and GEM ....................................................................... 62
5.3.4 Phosducin (Pdc) .................................................................................................................................................. 63
5.3.5 Ubiquitination controls Gα trafficking and degradation .................................................................................... 64
5.4 SMALL GTPASES ............................................................................................................................................................. 64
5.4.1 Introduction to small GTPases ............................................................................................................................ 64
5.4.2 Regulation of small GTPases ............................................................................................................................... 64
5.4.3 RAS and small GTPases ....................................................................................................................................... 65
5.5 CYCLASES AND SECOND MESSENGERS ................................................................................................................................... 66
5.5.1 Adenylyl cyclases ................................................................................................................................................ 66
5.5.2 Guanylyl cyclases ................................................................................................................................................ 70
5.5.3 Phosphodiesterases (PDEs) ................................................................................................................................. 71
H6: MEMBRANE LIPID DERIVED SIGNALING COMPOUNDS ............................................................................................ 73
Signal
and Cell
,
,H1: INTRODUCTION: CORE PRINCIPLES ............................................................................................................................2
1.1 GENERAL INTRODUCTION .................................................................................................................................................... 2
1.2 THE MAIN PRINCIPLES OF CELL SIGNALING ............................................................................................................................... 2
1.2.1 The main idea ....................................................................................................................................................... 2
1.2.2 Many extra components ....................................................................................................................................... 3
1.2.3 Signal transduction has different outcomes ......................................................................................................... 3
1.2.4 Points to keep in mind........................................................................................................................................... 3
1.3 CHANGE IS THE KEY ............................................................................................................................................................ 5
1.4 WHAT MAKES A GOOD SIGNAL? ............................................................................................................................................ 5
1.5 DIFFERENT WAYS IN WHICH CELLS SIGNAL TO EACH OTHER .......................................................................................................... 6
1.5.1 Electrical ............................................................................................................................................................... 6
1.5.2 Chemical ............................................................................................................................................................... 6
1.6 MECHANISMS OF CELL-TO-CELL SIGNALING ............................................................................................................................. 8
1.6.1 Direct cell-cell signaling ........................................................................................................................................ 8
1.6.2 Receptor-ligand signaling – cell-surface receptor ................................................................................................. 8
1.6.3 Vesicle-mediated signaling ................................................................................................................................. 11
1.7 SPATIAL AND TEMPORAL CONTROL IN CELL SIGNALING ............................................................................................................. 12
1.7.1 Interorganelle communication ........................................................................................................................... 12
1.7.2 Protein sorting and translocation ....................................................................................................................... 13
1.7.3 Protein modifications and organelle environments ............................................................................................ 14
1.7.4 Protein degradation ............................................................................................................................................ 15
1.7.5 Temporal and spatial control in cell signaling .................................................................................................... 15
1.7.6 Modular nature of signaling pathways .............................................................................................................. 15
1.8 DOMAINS AND MODULES .................................................................................................................................................. 16
1.9 CELLULAR SIGNALING IS NOT ONLY A MATTER OF CLASSICAL PROTEINS ......................................................................................... 17
1.9.1 The concept of signal amplification .................................................................................................................... 17
1.9.2 Feedback control and crosstalk .......................................................................................................................... 17
1.9.3 Non-proteins in cell signaling transduction ........................................................................................................ 17
H2: FIRST MESSENGERS ................................................................................................................................................. 18
2.1 SIGNALS AND RECEPTORS .................................................................................................................................................. 18
2.1.1 Terminology ........................................................................................................................................................ 18
2.1.2 Types of signals ................................................................................................................................................... 18
2.1.3 Eukaryotic organelles and cellular structures involved in cell signaling ............................................................. 18
2.2 CELL-TO-CELL COMMUNICATION ......................................................................................................................................... 19
2.3 SYNAPTIC TRANSMISSION .................................................................................................................................................. 20
2.3.1 The synapse ........................................................................................................................................................ 20
2.3.2 Neurotransmitters .............................................................................................................................................. 21
2.4 HORMONES .................................................................................................................................................................... 24
2.4.1 Specific hormones and their biological effects.................................................................................................... 24
2.4.2 Mode of action ................................................................................................................................................... 25
2.4.3 Hormonal regulation in insects ........................................................................................................................... 27
2.4.4 Plant hormones................................................................................................................................................... 28
H3: RECEPTORS .............................................................................................................................................................. 29
3.1 GENERAL INTRODUCTION AND PROPERTIES OF RECEPTORS ....................................................................................................... 29
3.2 LIGAND-RECEPTOR BINDING ............................................................................................................................................... 29
3.3 RECEPTOR SENSITIVITY AND RECEPTOR DENSITY...................................................................................................................... 31
3.3.1 Receptor sensitivity ............................................................................................................................................. 31
3.3.2 Receptor density ................................................................................................................................................. 32
3.4 RECEPTOR BINDING ASSAYS ................................................................................................................................................ 32
3.4.1 Steps in a receptor binding assay ....................................................................................................................... 33
3.4.2 Separation of bound and free ligand? ................................................................................................................ 33
3.5 DIFFERENT KINDS OF RECEPTORS, LIGANDS AND RESPONSES ..................................................................................................... 34
3.5.1 Different kinds of receptors ................................................................................................................................ 34
, 3.5.2 Different kinds of ligands .................................................................................................................................... 34
3.5.3 Different kinds of responses................................................................................................................................ 35
3.5.4 Molecular mechanisms behind signal transduction ........................................................................................... 35
3.6 INTRACELLULAR RECEPTORS ............................................................................................................................................... 36
3.6.1 Nuclear receptor family ...................................................................................................................................... 36
3.7 MEMBRANE RECEPTORS .................................................................................................................................................... 37
3.7.1 Ligand-gated ion channels .................................................................................................................................. 37
3.7.2 Single transmembrane receptors ........................................................................................................................ 38
3.7.3 Heptahelical protein receptors: G-protein coupled receptors (GPCRs) ............................................................... 39
3.8 LIGAND-RECEPTOR ASSAYS ................................................................................................................................................. 42
A. Scintillation proximity assay (SPA) ........................................................................................................................... 42
B. Cytosensor microphysiometer ................................................................................................................................. 42
C. Genetically engineered cell line ............................................................................................................................... 42
D. Intracellular calcium measurements with fluorescent dyes .................................................................................... 42
E. Co-expression with promiscuous Gα subunits ......................................................................................................... 42
F. Fluorescent imaging plate reader (FLIPR) ................................................................................................................ 42
G. Aequorin-based functional screening ...................................................................................................................... 42
H. Genetically encoded calcium or cAMP sensors ....................................................................................................... 42
I. cAMP sensor using YFP-Epac-RLuc (CAMYL) ............................................................................................................. 42
J. Bioluminescence resonance energy transfer (BRET) ................................................................................................. 42
K. NanoBiT protein-protein interaction system ............................................................................................................ 42
H4: PROTEIN PHOSPHORYLATION, KINASES AND PHOSPHATASES .................................................................................. 43
4.1 PHOSPHORYLATION .......................................................................................................................................................... 43
4.1.1 Covalent modification regulates enzyme activity ............................................................................................... 43
4.1.2 Phosphorylation? ................................................................................................................................................ 43
4.2 PROTEIN KINASES............................................................................................................................................................. 45
4.2.1 Serine/Threonine kinases.................................................................................................................................... 47
4.2.2 Tyrosine kinases .................................................................................................................................................. 55
4.3 PHOSPHATASES ............................................................................................................................................................... 57
H5: MODULATORS OF G-PROTEIN SIGNALING, CYCLASES AND PHOSPHODIESTERASES .................................................. 58
5.1 INTRODUCTION ............................................................................................................................................................... 58
5.1.1 GPCR and G-protein structure ............................................................................................................................ 58
5.1.2 Signaling and G protein subunits ........................................................................................................................ 58
5.2 REGULATORS OF GPCRS – RECEPTOR-LEVEL MODULATION....................................................................................................... 59
5.2.1 RAMPs – Receptor-Activity Modifying Proteins .................................................................................................. 59
5.2.2 GPCR desensitization .......................................................................................................................................... 59
5.2.3 G-protein receptor kinases (GRKs) ...................................................................................................................... 59
5.2.4 α- and β-arrestins ............................................................................................................................................... 60
5.3 REGULATORS OF G-PROTEINS – SUBUNIT-LEVEL MODULATION .................................................................................................. 61
5.3.1 Regulators of G-protein signaling (RGS) ............................................................................................................. 61
5.3.2 GoLoco motifs ..................................................................................................................................................... 61
5.3.3 Non-GPCR mediated activation of Gα proteins by GIV and GEM ....................................................................... 62
5.3.4 Phosducin (Pdc) .................................................................................................................................................. 63
5.3.5 Ubiquitination controls Gα trafficking and degradation .................................................................................... 64
5.4 SMALL GTPASES ............................................................................................................................................................. 64
5.4.1 Introduction to small GTPases ............................................................................................................................ 64
5.4.2 Regulation of small GTPases ............................................................................................................................... 64
5.4.3 RAS and small GTPases ....................................................................................................................................... 65
5.5 CYCLASES AND SECOND MESSENGERS ................................................................................................................................... 66
5.5.1 Adenylyl cyclases ................................................................................................................................................ 66
5.5.2 Guanylyl cyclases ................................................................................................................................................ 70
5.5.3 Phosphodiesterases (PDEs) ................................................................................................................................. 71
H6: MEMBRANE LIPID DERIVED SIGNALING COMPOUNDS ............................................................................................ 73
