Cell Biology Lecture 9 Notes Basic Principles of cell signaling and GPCR system

Transcription

1 Cell Biology Lecture 9 Notes Basic Principles of cell signaling and GPCR system Basic Elements of cell signaling: Signal or signaling molecule (ligand, first messenger) o Small molecules (epinephrine, acetylcholine, steroids), peptides, hormones, & light secreted by signaling cells o First messenger molecule brings first message to the cell to do something Receptors recognize the signal o Cell-surface receptors o Intracellular receptors Intracellular signaling and effector proteins o Receptors activated and instigate a sequence of events in the cell o G proteins, protein kinases (phosphorylate amino acids serine, tyrosine, thymine) and phosphates Second messengers o Small molecules such as Ca2+, camp, cgmp, IP3, DAG, NO All cells receive and respond to signals from their environment cells and environment must communicate Four forms of intercellular signaling: Signaling molecules can be delivered to the cell by different mechanisms Endocrine = signaling to distant cells o Signaling molecules are synthesized & secreted by signaling cells (endocrine cells) & are transported through circulatory system to target cells o The hormone generally refers to signaling molecules that mediate endocrine signaling Paracrine = signaling to nearby cells

2 o The signaling molecules released by a cell affect only those target cells in close proximity o E.g. neurotransmitters Autocrine = signaling to cell itself o Cells respond to substances that they themselves release o Have receptor & recognize signal & develop response to signal molecule o E.g. tumour cells express receptors for different growth factors Signaling by PM-attached proteins o Signal molecules are membrane attached proteins o Make a cell complex not like a cell-adhesion cell complex just based on interactions between signal molecule and the cell Ligand-receptor interactions: Binding specificity is based on the molecular complementarity (molecular recognition) between the interacting surfaces of a receptor (binding interface) and ligand (non-covalent forces) o Surfaces that interact tightly = complementary to each other Interactions trigger a conformational change in the receptor becomes active Very often signaling molecules (ligands) induce receptor dimerization The Dissociation Constant: R + L RL at equilibrium we have a simple equilibrium-binding equation: K d = [R][L]/[RL] o K d is the measure of affinity of a receptor to its ligand o K d is ligand concentration required to bind 50% of cell surface receptors o The lower the K d, the lower the ligand concentration required to occupy 50% of the cell surface receptors Ligand binding to a receptor usually can be viewed as a simple reversible reaction Nuclear-receptor superfamily: Specific type of receptors that are NOT located in the membrane cytoplasm or nucleus Transcription factors that are activated once they bind their appropriate ligand Focus only on the domains of the general primary structure Lipid-soluble molecules/ hormones bind to intracellular receptors which constitute the nuclear receptor superfamily of transcription factors o Steroid hormones are extracellular & are used by intracellular receptor to mediate cell response are synthesized by adrenal glands

3 Ligand binding domain binds to specific hormones DNA binding domain binding the specific DNA sequence (response element) Variable region significant difference between transcription factors have one of several activation domains within the variable region Gene activation by a nuclear receptor: GR is a steroid hormone can diffuse through PM and get inside the cell because is lipid soluble o Is a potent anti-inflammatory/ immuno-suppressive reagent & others The characteristic nucleotide sequences of the DNA sites that bind nuclear receptors are called response elements o The receptor binds to a response element of the target gene and stimulate pre-initiation complex assembly required for transcription (mrna synthesis) In the absence of hormone, the receptor in trapped in the cytoplasm by inhibitor proteins (e.g. HSP90) o Hormone binding to a nuclear receptor releases the inhibitor protein, allowing the receptor to enter the nucleus Binding to ligand binding domain changes conformation which releases the inhibitor from the domain ligand can diffuse to the nucleus and interact with the response element induce transcription of specific gene

4 Signaling by cell-surface receptors: Converting extracellular signals to intracellular responses = signal transduction Termination of the cellular response is caused by intracellular signaling molecules that inhibit receptor function & by removal of the extracellular signal Short-term cellular responses associated with changes in the activity & functions of specific enzymes & other proteins that pre-exist in the cell Long-term cellular responses associated with changes in amounts of specific proteins produced by a cell, most commonly by modification of transcription factor activation that stimulate or repress gene expression G protein-coupled receptor system: Requires a receptor with 7 membrane-spanning domains Requires a coupled trimeric G proteins associates with receptor Requires a membrane bound effector protein eventually mediates the effect of binding of specific ligand to receptor Requires a second messenger in many GPCR pathways Responsible for many different functions o Vision, smell o Glycogen metabolism o Development o Arteriole smooth muscle relaxation o Exploited by pathogens (whooping cough & cholera) o Exploited by drug companies o Exploited by drug dealers (meth, heroin, ecstasy etc.) GPCRs is the most numerous class of receptors in animal cells G protein-coupled receptors: All have same structure have 7 transmembrane domains (alpha helices) which are labeled H1-H7 There are many subfamilies of GPCR which are conserved through evolution Cytoplasmic segments binds G-proteins Exoplasmic/extracellular segments binding of ligands

5 Trimeric G-protein: The GTPase superfamily of proteins; G refers to ability to bind guanine nucleotides (GDP or GTP) Hetero-trimer of three subunits: G, G, and G Gand G are lipid-anchored proteins at the cytoplasmic face of the plasma membrane G-GDP is inactive, G-GTP is active G is central part of complex subunit is active or inactive Function in signal transduction by coupling ligand-bound receptors to specific effector molecules How to demonstrate receptor-mediated dissociation of G and G - FRET: FRET (fluorescence resonance energy transfer) based on transfer of fluorescent energy if they are located close to each other Cells are transfected with genes encoding two fusion proteins: 1) G-alpha fused to cyan fluorescent protein (CFP) 2) G-beta fused with yellow fluorescent protein (YFP) CFP and YFP are mutant forms of GFP When CFP and YFP are nearby, as in resting G-alpha/ G-beta, gamma complex, fluorescence energy transfer can occur between CFP and YFP

6 Irradiation of resting cells with 440 nm of light (which directly excites CFP but not YFP) causes emission of 527 nm of (yellow) light, characteristic of YFP Once subunits dissociate, irradiation of cells at 440 nm causes emission of 490 nm of light (cyan), characteristic of CFP Complex of G-alpha GTP makes effector molecule active (this state does not last a long time) GTP will be hydrolyzed and released from this complex alpha subunits will become inactive and can bind GDP Signals are most commonly transduced by G-alpha - a GTPase switch protein that alternates between an active ( on ) state with bound GTP and inactive ( off ) state with GDP The beta and gamma subunits, which remain bound together, occasionally transduce signals Once you add hormone in the presence of specific stimulus, this signal molecule will bind to the receptor and activate the receptor due to change in conformation change in receptor conformation allows it to bind alpha subunits dissociation of GDP from nucleotide binding site of subunits

7