MCB110-Spring 2008-NogalesSignaling 15 CELL SIGNALINGI IntroductionA. Types of SignalingB. Types of ReceptorsC. Other Conserved FunctionsII G Protein-coupled ReceptorsA. Heterotrimeric G proteinsB. G-protein coupled receptorsC. Second messenger cAMP: Glucose mobilizationD. Lipid-derived Second MessengersE. Calcium SignalingIII Receptor Tyrosine KinasesA. Insulin ReceptorB. General RTKs, Ras and the MAP kinase cascadeIV Integrin SignalingV Cell Signaling and ApoptosisVI Relationships between Signaling PathwaysSuggested Reading: Lodish, Chapter 13; Chapter 14, 14.3 to 14.5Alberts, Chapter 15MCB110-Spring 2008-NogalesSignaling 2Essential to the survival of every cell is tomonitor the environment and to respond toexternal stimuli. For most cells this includesappropriate communication with neighboringcells. Cell signaling (or signal transduction)involves:• Detection of the stimulus (in mostcases a molecule secreted by anothercell) on the surface of the plasmamembrane.• Transfer of the signal to thecytoplasmic side.• Transmission of the signal to effectormolecules and down a signaling pathwaywhere every protein typically changesthe conformation of the next down thepath, most commonly byphosphorylation (by kinases) ordephosphorylation (by phosphatases).The final effect is to trigger a cell’sresponse, such as the activation ofgene transcriptionI INTRODUCTION TO SIGNALING PATHWAYSMCB110-Spring 2008-NogalesSignaling 3MCB110-Spring 2008-NogalesSignaling 4I A – Types of SignalingCells communicate by means of extracellular signaling molecules that are producedand released by signaling cells. These molecules recognize and bind to receptors onthe surface of target cells where they cause a cellular response by means of a signaltransduction pathway.Depending on the distance that the signaling molecule has to travel, we can talk aboutthree types of signaling:In endocrine signaling hormones are produce by an endocrine gland and sent through the bloodstream to distant cells.Hormones can be: small lipophilic molecules that diffuse through the cell membrane to reachcytosolic or nuclear receptors. Examples are progesterone and testosterone, as well as thyroidhormones. They generally regulate transcription; or water soluble molecules that bind to receptorson the plasma membrane. They are either proteins like insulin and glucagons, or small, chargedmolecules like histamine and epinephrine.MCB110-Spring 2008-NogalesSignaling 5In paracrine signaling the signalingmolecule affects only target cells inthe proximity of the signaling cell. Anexample is the conduction of anelectric signal from one nerve cell toanother or to a muscle cell. In this casethe signaling molecule is aneurotransmitter.In autocrine signaling cells respond tomolecules they produce themselves.Examples include many growth factors.Prostaglandines, lipophilic hormones thatbind to membrane receptors, are often usedin paracrine and autocrine signaling. Theygenerally modulate the effect of otherhormones.Once a signaling molecule binds to its receptor it causes a conformational change in it that results in acellular response. The same ligand can bind to different receptors causing different responses (e.g..acetylcholine). On the other hand, different ligands binding to different receptors can produce thesame cellular response (e.g. glucagon, epinephrine).MCB110-Spring 2008-NogalesSignaling 6I B – Types of ReceptorsThere are a number of receptor classes that are used in different signaling pathways.The two more predominant are:The conformational change in the receptor upon ligand binding activates a G protein,which in turns activates an effector protein that generates a second messenger.MCB110-Spring 2008-NogalesSignaling 7These receptors have a catalytic activity that is activated by binding of the ligand. Anexample are tyrosine-kinase receptors. Binding of an often dimeric ligand inducesdimerization of the receptors that leads to cross phosphorylation of the cytosolicdomains and phosphorylation of other proteins.MCB110-Spring 2008-NogalesSignaling 8Identification and Purification of Cell-Surface ReceptorsHormone receptors are difficult to identify and purify because they are present invery low abundance and they have to be solubilized with nonionic detergents. Giventheir high specificity and high affinity for their ligands, the presence of a certainreceptor in a cell can be detected and quantified by their binding to radioactively-labeled hormones. The binding of the hormone to a cell suspensions increases withhormone concentration until it reaches receptor saturation. Specific binding isobtained by measuring both the total and the non-specific binding (which is obtainedby using a large excess on unlabeled hormone).MCB110-Spring 2008-NogalesSignaling 9The receptor can be purified insome cases by means of affinitychromatography: the hormone islinked to agarose beads. Crude,solubulized membranes arepassed through a column withthese beads, which will retain onlytheir specific receptor. Thereceptor is later release bypassing excess hormone throughthe column. A single pass canproduce a 100,00 fold enrichment.MCB110-Spring 2008-NogalesSignaling 10In many cases, however, the number of receptor on the cell surface is to low to usechromatography. An alternative is to produce recombinant receptor protein.A plasmid cDNA library from the cellsexpressing the receptor is screen bytransfecting the cloned cDNAs into cells thatnormally do not express the receptor. Cellsthat take up the cDNA encoding the receptorare detected by their binding to fluorescenceor radioactively-labeled hormone. The cDNAcan then be sequenced and used produce largeamounts of recombinant protein.MCB110-Spring 2008-NogalesSignaling 11MCB110-Spring 2008-NogalesSignaling 12I C – Other Conserved FunctionsThe three main classes of intracellular signaling proteins are:G proteins (GTPase switch proteins) - These proteins change between an activeconformation when bound to GTP, and an inactive conformation when bound to GDP.IN the absence of a signal they are bound to GDP. Signal results in the release ofGDP and the binding of abundant GTP. After a short period of time they hydrolyseGTP and come back to their “off” state.MCB110-Spring 2008-NogalesSignaling 13Protein Kinases - Upon activation they add phosphate groups to themselves and/orother proteins at either serine/threonine, or at tyrosine residues. Their activity canbe regulated by second messengers, interaction with