Cellular Communication 3 2024.pdf

ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE Cellular Biology & Homeostasis CELLULAR COMMUNICATION Part 3 VP 2024 Clara Camargo, DVM ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE LEARNING OBJECTIVES 1. Understand how the G protein βꝩ subunits participate in cellular signaling 2. Describe the GPCR-P...

ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE Cellular Biology & Homeostasis CELLULAR COMMUNICATION Part 3 VP 2024 Clara Camargo, DVM ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE LEARNING OBJECTIVES 1. Understand how the G protein βꝩ subunits participate in cellular signaling 2. Describe the GPCR-PIP2 pathway and the second messengers associated with it 3. Differentiate the effects and mechanism of action of the different second messengers discussed (cAMP, Ca+2, DAG, IP3) 4. Describe the Receptor Tyrosine Kinases (RTKs) transduction mechanism 5. Describe the functioning of gated ion channels (ionotropic receptors) 6. Understand the transduction mechanism of intracellular receptors 7. Describe the receptor mediated endocytosis ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE RECALL: GPCR cAMP pathway GAP PKA activation ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE cAMP acts as a Second Messenger for several regulatory molecules.  Protein Kinase A (PKA) → cAMP binds to regulatory site and activates PKA: activates enzymes involved in:  fat mobilization and  cortisol synthesis can also move into the nucleus, where it phosphorylates CREB (cAMP response element binding protein)  This alters the expression of specific genes regulated by [cAMP] in the cell  By binding to specific genes on the DNA and either stimulating or inhibiting their transcription ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE https://themedicalbiochemistrypage.org/lipolysis-and-theoxidation-of-fatty-acids Activation of enzymes involved in fat mobilization Glucagon (first messenger) → Adipocytes  Gs activation of AC  increase [cAMP]  stimulates PKA  PKA activates enzymes involved in fat mobilization (hormone sensitive lipase) ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE Activation of CREB via activated PKA Extracellular Intracellular ↑ [cAMP] ↑ activated PKA cAMP Response Element Binding Protein (CREB) ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE Ca++ RELEASE: Can be triggered by cAMP pathway  via activation of PKA and cell surface Ca++ channels Epinephrine/Adrenaline ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE G proteins do not act exclusively by regulating the activity of effector enzymes They can directly activate or inactivate ion channels in the plasma membrane → through the action of the βꝩ subunits Ligand-gated ion channel Gi inhibits Adenylyl cyclase while βγ subunits bind to K channels, that will open making depolarization of the cells more difficult GPCR Can alter the ion permeability and electrical excitability  E.g., Acetylcholine released by the vagus nerve reduces heart rate and stimulates smooth muscle contraction ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE Another broad class of GPCRs are coupled to a Gq protein and activates a phospholipase C (PLC)  The enzyme PLC is specific for a membrane phospholipid: PIP2 (phosphatidylinositol 4,5-bisphosphate) DAG PIP2 IP3 ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE 1. Signal hormone binding to the specific 7. Many enzymes activated by PKC affect receptor A cytoskeletal proteins, nuclear proteins, and other enzymes (cellular response) 2. Activates Gq protein 3. Which activates the PIP2-specific PLC 4. This activation catalyzes the production of two powerful second messengers: diacylglycerol (DAG) and inositol 1,4,5trisphosphate (IP3) 5. IP3 is hydrophilic, diffuses to ER and binds to specific IP3-gated Ca2+ channels causing them to open and release stored Ca2+ to the cytosol 6. ↑ [Ca2+ ] in combination with DAG activates Protein Kinase C (PKC) ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE Ca2+ serves as second messenger triggering different intracellular responses: – Exocytosis: neurons (neurotransmitter vesicles), mast cells (histamine granules), endocrine cells (hormones) – Contraction in muscles – Cytoskeletal movement in amoeba  Cytosolic [Ca2+ ] are kept very low by action of Ca2+ pumps (i.e., Na+/Ca+2 exchanger) in ER, mitochondria, plasma membrane  Hormonal, neural or other stimuli causes influx of Ca2+ into the cell either through special Ca2+ channels in the membrane, or release sequestered Ca2+ from ER or mitochondria  Rises in intracellular [Ca2+ ] will then cause cytosolic responses ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE Changes in intracellular [Ca2+ ] are sensed by Calmodulin (CaM)  Calmodulin (CaM) - Ca2+- binding proteins that regulate many Ca2+ – dependent enzymes/proteins CaM has 4 high-affinity Ca2+–binding sites At a certain threshold [Ca2+ ], binding of Ca2+ to calmodulin causes a conformational change Calmodulin bound to Ca2+ can bind to and modulate the activity of different proteins  i.e., CaM-dependent kinases and other enzymes  In smooth muscle → CaM binds to the myosin light chain kinase → myosin head is activated → stimulates cross-bridge cycling ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE MAJOR TYPES OF RECEPTORS and SIGNAL TRANSDUCERS From: Lehninger Principles of Biochemistry ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE Kinase → enzymes → phosphorylation of proteins via covalent bonds (adding phosphate groups using ATP) Phosphorylation → functional change of the target protein  changing enzyme activity  cellular location  association with other proteins Phosphorylation can occur by:  autophosphorylation  binding of activator/inhibitor proteins (kinases)  Phosphorylation of proteins regulate several cellular pathways! ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE 2 MAIN TYPES Serine/Threonine Kinases Phosphorylate serine and threonine aa in their targets EX: MAP kinase (mitoses, osmotic stress, cellular differentiation) Tyrosine Kinases Phosphorylates proteins on tyrosine amino acid residues EX: INSULIN RECEPTOR ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE RTKs are a large family of membrane receptors with intrinsic protein kinase activity (autophosphorylate) They transduce extracellular signals by a different mechanism to GPCRs RTKs have:  ligand-binding domain on the extracellular face of the membrane and  enzyme active site on the cytoplasmic face, connected by a single transmembrane segment The cytoplasmic domain is a protein kinase that phosphorylates TYR residues  Examples: receptors for insulin and epidermal growth factors ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE Example: Insulin Receptor ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE Major regulatory mechanism is receptor sequestration and downregulation (receptor endocytosis and degradation) Other ways the receptor can be regulated:  Modulation of the phosphorylation state of RTKs  Antagonist drugs can inhibit the biological responses mediated by the receptor ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE Certain cells are ‘excitable’, they can detect an  Excitability depends on ion channels: signal external signal, convert it into an electrical signal transducers which form a regulated path for (change in membrane potential) and pass it on movement of inorganic ions (Na+, K+, Ca2+,Cl-) across the plasma membrane in response to  Excitable cells are central in various stimuli Nerve conduction Muscle contraction Hormone secretion Sensory and learning processes  These channels are gated and must be activated by Change in transmembrane electrical potential (voltage) or Binding of specific ligand (i.e. neurotransmitter) ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE Nicotinic acetylcholine (ACh) receptor is a ligand-gated ion channel It mediates the passage of an electrical signal at some types of synapses and at the neuromuscular junction (between motor neuron and muscle fiber) Their signal causes depolarization of membrane, triggering skeletal muscle contraction ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE INTRACELLULAR RECEPTORS Lipophilic signals are carried by transport proteins in the blood plasma from which they dissociate before entering a target cell These small hydrophobic signal molecules diffuse directly across the plasma membrane of target cells Bind to intracellular receptors (in the cytoplasm OR in the nucleus) that are transcription regulators  EX: Steroid hormones, thyroid hormones, retinoid and vitamin D ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE INTRACELLULAR RECEPTORS INTRACELLULAR RECEPTORS ARE RECEPTORS AND EFFECTORS OF THE SIGNAL Ligands bind to their respective intracellular receptor proteins and alter the ability of these proteins to control the transcription of specific genes ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE RECEPTOR MEDIATED ENDOCYTOSIS Is initiated when receptors become activated by ligand Membrane associated protein CLATHRIN is essential for this mechanism  Ex: endocytosis of cholesterol via LDL