Receptorligand interactions cell signaling adhesion motility cell migration

"Receptor-ligand interactions - cell signaling, adhesion, motility, cell migration" Patricia Zuk, Ph. D Research Director Regenerative Bioengineering and Repair (REBAR) Lab Department of Surgery David Geffen School of Medicine at UCLA

No cell lives in isolation • survival depends on an elaborate intercellular communication network that coordinates growth, differentiation and metabolism • cells adjacent to one another frequently communicate through cell-cell contact • other forms of communication cover larger distances = extracellular signaling molecules

Extracellular Signalling • • signaling molecules are released by signaling cells the signal is called the ligand binds to its specific receptor on a target cell this ligand-receptor interaction induces a conformational or shape-change in the receptor • produces a specific response - called the cellular response • can include a vast array of compounds – e. g. small amino acid derivatives, small peptides, proteins

Cell-to-cell communication by extracellular signaling usually involves six steps • • (1) synthesis of the signaling molecule by the signaling cell (2) release of the signaling molecule by the signaling cell (3) transport of the signal to the target cell (4) detection of the signal by a specific receptor protein – receptor-ligand specificity • (5) a change in cellular metabolism, function, or development = cellular response – triggered by the receptor-ligand complex – specific to the ligandreceptor complex • (6) removal of the signal, which usually terminates the cellular response – degredation of ligand

Signaling molecules operate over various distances in animals -extracellular signaling can occur over: 1. large distances or endocrine signaling – signaling molecules are called hormones - act on target cells distant from their site of synthesis -usually carried through the bloodstream 2. short distances or paracrine signaling – affects target cells within proximity to the cell that synthesized the molecule -usually mediated by neurotransmitters and some growth factors

Signaling molecules operate over various distances in animals -extracellular signaling can occur over: 3. no distance or autocrine signaling – the signal feeds-back and affects itself -action of many growth factors -these compounds generally act on themselves to regulate proliferation -seen frequently in tumor cells -many compounds can act through two or even three types of cell signaling e. g. growth factors (e. g. EGF) – paracrine and autocrine and endocrine -epinephrine – endocrine and paracrine

Circulating & Local Hormones • Circulating hormones – act on distant targets – travel in blood – endocrine hormones • Local hormones – paracrine hormones & autocrine hormones

Hormones • two types – lipid soluble – water soluble

Lipid-Soluble Hormones -lipid-soluble hormones can easily enter a cell by diffusing through the plasma membrane -PROBLEM: how do they travel in the water-based blood? ? -SOLUTION: they are carried by carrier-proteins -these hormones then enter their target cell where they result in a specific cellular effect or response

Water-soluble Hormones -water soluble hormones can easily travel within the blood -PROBLEM: how do they enter a cell and result in a cellular response? ? -SOLUTION: binding to specific cell-surface receptors -this binding activates the receptor and results in a series of cellular events called the second messenger system

Lipid-soluble Hormones • Steroids – lipids derived from cholesterol in SER – different functional groups attached to core of structure provide uniqueness – interact with specific intracellular receptors (within the cell) to turn specific genes on or off – effective for hours or days • Thyroid hormones – tyrosine ring plus attached iodines are lipid-soluble – activate enzymes involved in the catabolism of fats and glucose – help set our basal metabolic rate • Retinoids – vitamin A derivatives – have dramatic effects on proliferation and differentiation plus cellular death (i. e. apoptosis)

Water-soluble Hormones • Amino acid derivatives, small peptides and protein hormones – modified amino acids or amino acids put together • serotonin, melatonin, histamine, epinephrine – larger peptide hormones • insulin and glucagon • Eicosanoids – derived from arachidonic acid (fatty acid) – prostaglandins or leukotrienes – prostaglandins despite being lipidphilic – bind to cell surface

Action of Lipid-Soluble Hormones • Hormone diffuses through phospholipid bilayer & into cell • Binds to receptor turning on/off specific genes • New m. RNA is formed & directs synthesis of new proteins • New protein alters cell’s activity

Action of Water-Soluble Hormones • Can not diffuse through plasma membrane • Hormone receptors are integral membrane proteins – act as first messenger • Receptor protein activates G-protein in membrane • G-protein activates adenylate cyclase to convert ATP to c. AMP in the cytosol • Cyclic AMP is the 2 nd messenger • Activates kinases in the cytosol to speed up/slow down physiological responses • Phosphodiesterase inactivates c. AMP quickly • Cell response is turned off unless new hormone molecules arrive

Cell-surface receptors belong to four major classes • • GPCRs are involved in a range of signaling pathways, including light detection, odorant detection, and detection of certain hormones and neurotransmitters Many different mammalian cell-surface receptors including GPCRs are coupled to a trimeric signal-transducing G protein – made of an alpha, beta and gamma subunit complex Ligand binding activates the receptor, which activates the G protein, which activates an effector enzyme to generate an intracellular second messenger – e. g. adenylyl cyclase – converts ATP to c. AMP depending on regulation at the effector enzyme – this pathway can be either activated or inhibited – by the type of G protein activated by the hormone-receptor complex – Gs proteins result in stimulation of the effector enzyme – Gi proteins result in inhibition of the effector enzyme adenylyl cyclase (AC)

Four classes of cell-surface receptors -ligand binding changes the confirmation of the receptor so that specific ions flow through it -the resultant ion movement alters the electric potential across the plasma membrane -found in high numbers on neuronal plasma membranes e. g. ligand-gated channels for sodium and potassium -also found on the plasma membrane of muscle cells -binding of acetylcholine results in ion movement and eventual contraction of muscle

-lack intrinsic catalytic activity -binding of the ligand results in the formation of a receptor dimer (2 receptors) -this dimer than activates a class of protein called tyrosine kinases -this activation results in the phosphorylation of downstream targets by these tyrosine kinases (stick phosphate groups onto tyrosines within the target protein) -receptors for cytokines such as XXXX, interferons (XXXXXXX)

Signal transduction Cascade -also called receptor tyrosine kinases OR ligand-triggered protein kinases -similar to tyrosine-linked receptors - ligand binding results in formation of a dimer -BUT: they differ from tyrosine-linked receptors – intrinsic catalytic activity -means that ligand binding activates it and the activated receptor acts as a kinase -recognize soluble or membrane bound peptide/protein hormones that act as growth factors e. g. NGF, PDGF, insulin -binding of the ligand stimulates the receptor’s tyrosine kinase activity, -results in phosphorylation of multiple amino acid residues within its target – such as serine and threonine residues -this phosphorylation activates downstream targets -its targets are generally other protein kinases –which phosphorylate their own downstream targets (other kinases? ? )

Signal transduction cascades Signal • -the successive phosphorylation/activation of multiple kinases results in a cascade of phosphorylation/activation • -this cascade is frequently called a signal-transduction cascade • -this cascade eventually leads to a specific cellular response • e. g. changes in cell physiology and/or patterns of gene expression • -RTK pathways are involved in regulation of cell proliferation and differentiation, promotion of cell survival, and • modulation of cellular metabolism p KINASE #1 p KINASE #2 p KINASE #3 TARGET EFFECT p

Second messengers • produced by the activation of GPCRs and RTKs • Hormone stimulation of Gs protein-coupled receptors leads to activation of adenylyl cyclase and synthesis of the second messenger c. AMP – most commonly studied second messenger – c. AMP does not function in signal pathways initiated by RTKs – c. AMP and other second messengers activate specific protein kinases (c. AMPdependent protein kinases or PKAs) • c. AMP has a wide variety of effects depending on the cell type and the downstream PKAs and other kinases – in adipocytes, increased c. AMP activates a PKA that stimulates production of fatty acids – in ovarian cells another PKA will respond to c. AMP by increase estrogen synthesis • second messenger systems allow for amplification of an extracellular signal – one epinephine molecule can bind one GPCR – this can result in the synthesis of multiple c. AMP molecules which can go on to activate and amplified number of PKAs • a blood level as low as 10 -10 M epinephrine can raise blood glucose levels by 50%

Second messengers • other second messengers include: – IP 3 and DAG – breakdown products of phosphotidylinositol (PI) • produced upon activation of multiple hormone receptor types (GPCRs and RTKs) – calcium – IP 3 production results in the opening of calciumchannels on the plasma membrane of the ER – release of calcium • a rise in calcium in pancreatic beta cells triggers the exocytosis of insulin • a rise in intracellular calcium also triggers contraction of muscle cells • much study has been done on the binding of calcium to a protein called calmodulin and the effect of this complex on gene expression

MAP kinase pathways • • • best characterized signal transduction pathway activation of RTKs by growth factors, hormones etc…. . result in activation of an adaptor protein called Ras GTPase ras induces a kinase signal cascade that starts with a kinase called rac and culminates in activation of a MAP kinase (MAPK) in between are a series of kinases that are part of the cascade MAPK activation results in translocation into the nucleus and phosphorylate many different proteins, including transcription factors that regulate gene expression

MAPK kinase paths Ø Stress, cytokines, hormones & mitogens signal through cdc 42/rac Øcdc 42/rac – then activates one of three MAPK paths: Stress Cytokines Stress Hormones Cytokines Mitogens Hormones Øp 38 MAPK: stress response & apoptosis (MAPKAP- 2, HSP 27) cdc 42/rac ØJNK: stress response & proliferation (jun) Ø Activation of ras/MEK/ERK path: proliferation & grb 2 differentiation MAPK kinase transcription factors (nucleus) MEKKs MEK 3/6 JNKK 1/2 p 38 MAPKAP-2 HSP 27 JNK 1/2 ATF elk jun elk ras raf-1 MEK 1/2 ERK 1/2 elk RSK fos sos

Common signaling pathways are initiated by different receptors in a class • • • The effects of activation of GPCRs and RTKs is more complicated than a simple step-by-step cascade Stimulation of either GPCRs or RTKs often leads to production of multiple second messengers, and both types of receptors promote or inhibit production of many of the same second messengers in addition, RTKs can promote a signal transduction cascade that eventually acts on the same target as the GPCR therefore the same cellular response may be induced by multiple signaling pathways by distinct mechanisms Interaction of different signaling pathways permits fine-tuning of cellular activities

Integrating Cells into Tissues: Cell-Cell Adhesion and Communication -a key event in the evolution of multicellularity is the ability for cells to adhere to one another and be able to communicate with each other -evolved a series of cell-adhesion molecules or CAMs that allow interaction with each other and with the surrounding extracellular matrix (ECM) -this results in coordinated functioning of tissues -HOW? ? -these interactions result in the activation of specific signal transduction cascades eventually resulting in the desired cellular effect -therefore the physical interaction of CAMs with the ECM can turn pathways on or off – cellular effect e. g. cellular interactions with the adhesion protein b 1 -integrin can result in activation of the MAPK cascade

-various classes of CAMs found on cells 1. cadherins – cell-cell adhesion -calcium dependent e. g, E-cadherin, P-cadherin 2. Ig superfamily of CAMs – cell-cell adhesion e. g. N-CAM, V-CAM -calcium-independent -some are found enriched on specific cell types – N-CAM 3. Integrins – major cell-matrix adhesion molecule e. g. a 1 integrin, b 1 integrin

Cell-Cell adhesion: Cadherin-containing junctions 1. adherens junction – continuous band of cadherins found in epithelial cells -connects cells tightly and interact with the actin portion of the cytoskeleton 2. desmosomes – also found in high #s in epithelial tissues -cells attach via cadherins – connect to protein “plaques” that attach to the plasma membrane and to intermediate filaments within the cytoskeleton -cadherins are a family of calcium-dependent CAMs -major molecules of cell-cell adhesion (homophilic) -over 40 different are known e. g. E or epithelial cadherin N or neural cadherin -tissues have specialized junctions made of cadherins 1. Adherens junction 2. Desmosome

Cell Matrix • three components to the ECM – insoluble collagens – structural framework of the ECM, provides strength and resiliency – proteoglycans – cushions cells – adhesive matrix proteins – bind these components to receptors on the cell surface – different combinations result in unique ECM compositions which can directly affect the activity of the cells – the interaction of ECM components with specific CAMs (i. e. ECM receptors) can turn certain signaling paths ON or OFF – also the ECM is capable of sequestering growth factors and hormones by binding them and making them unavailable to turn cellular signaling on or off

Cell-matrix interactions: integrins • • integrins allow connection between the extracellular matrix with the cytoskeleton of the cell also can mediate cell-cell interactions (weak) made of an alpha and a beta subunit – 17 types of a chains, 8 types of b chains these ab complexes act as receptors to specific matrix components – e. g. a 1 b 1 – collagen and laminin – a 5 b 1 - fibronectin • • • cells will express multiple integrin types attachment can be regulated by up- or down-regulated expression of these integrins can also form very specific adhesive junctions – focal adhesions (FN to actin) – complex of more than 20 proteins • can increase and decrease based on physical stress – hemidesmosomes (intermediate filaments to collagens and laminins) – epithelial cells • activation of these integrins can promote cell signaling • e. g. formation and activation of focal adhesions triggers a cascade initaited by a Focal Adhesion Kinase (FAK) – modulates cell growth and motility

Cell-Cell Communication: Gap Junctions -almost all cells have specialized junctions that allow the free passage of materials (e. g. signaling molecules) back and forth -these junctions = gap junctions -made of a channel protein called connexon -connexons interact to form channels between two cells -one important compound small enough to traverse this junction is the second messenger c. AMP -also allows passage of calcium ions and other ions crucial to signaling (sodium, phosphate) -these gaps are not free-swinging gates -they actually open and close in response to ion concentrations thereby restricting the flow of too many ions -they also exclude materials based on size

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