"Receptor-ligand interactions - cell signaling, adhesion, motility,

cell migration"

Patricia Zuk, PhDResearch Director

Regenerative Bioengineering and Repair (REBAR) LabDepartment 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• 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 ligand-

receptor 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 proximityto the cell that synthesized the molecule-usually mediated by neurotransmitters and some growthfactors

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 toregulate proliferation-seen frequently in tumor cells

-many compounds can act through two or even three types of cell signalinge.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 thewater-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 calledthe 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 receptors

Action of Lipid-Soluble Hormones

• Hormone diffuses through phospholipid bilayer & into cell

• Binds to receptor turning on/off specific genes

• New mRNA 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 cAMP in the cytosol

• Cyclic AMP is the 2nd messenger• Activates kinases in the cytosol to

speed up/slow down physiological responses

• Phosphodiesterase inactivates cAMP 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 cAMP• 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 plasmamembrane

-found in high numbers on neuronal plasma membranese.g. ligand-gated channels for sodium and potassium

-also found on the plasma membrane of muscle cells-binding of acetylcholine results in ion movement andeventual 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 withinthe target protein)

-receptors for cytokines such as XXXX, interferons (XXXXXXX)

-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 transductionCascade

Signal transduction cascades

• -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

Signal

KINASE #2

KINASE #3

TARGET

EFFECT

KINASE #1pp

pp

pp

pp

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 cAMP– most commonly studied second messenger– cAMP does not function in signal pathways initiated by RTKs– cAMP and other second messengers activate specific protein kinases (cAMP-

dependent protein kinases or PKAs)• cAMP has a wide variety of effects depending on the cell type and the

downstream PKAs and other kinases– in adipocytes, increased cAMP activates a PKA that stimulates production of

fatty acids– in ovarian cells another PKA will respond to cAMP 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 cAMP molecules which can go on to activate and amplified number of PKAs

• a blood level as low as 10-10M epinephrine can raise blood glucose levels by 50%

Second messengers• other second messengers include:

– IP3 and DAG – breakdown products of phosphotidylinositol (PI)• produced upon activation of multiple hormone receptor types

(GPCRs and RTKs)– calcium – IP3 production results in the opening of calcium-

channels 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

StressCytokines

StressHormonesCytokines

MitogensHormones

cdc42/rac

MEKKs

JNKK 1/2

JNK 1/2

elkjunATFMAPKAP-2

HSP27

MEK 3/6

MEKKs

p38MAPK

ATFelk

cdc42/rac – then activates one of cdc42/rac – then activates one of three MAPK paths:three MAPK paths:

Activation of Activation of ras/MEK/ERK ras/MEK/ERK path: path: proliferation & differentiationproliferation & differentiation

p38MAPK:p38MAPK: stress response & stress response & apoptosis (MAPKAP-2, HSP27)apoptosis (MAPKAP-2, HSP27)

Stress, cytokines, hormones & Stress, cytokines, hormones & mitogens signal through mitogens signal through cdc42/raccdc42/rac

raf-1

MEK 1/2

ERK 1/2

elkRSK

rasgrb2

sos

fos

JNK:JNK: stress response & proliferation stress response & proliferation (jun)(jun)

transcription factors (nucleus)

MAPK kinase

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 oneanother and be able to communicate with each other-evolved a series of cell-adhesion molecules or CAMs that allow interaction witheach 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 oroff – cellular effect

e.g. cellular interactions with the adhesion protein 1-integrin can result in activation of the MAPK cascade

-various classes of CAMs found on cells1. cadherins – cell-cell adhesion

-calcium dependente.g, E-cadherin, P-cadherin

2. Ig superfamily of CAMs – cell-cell adhesione.g. N-CAM, V-CAM-calcium-independent-some are found enriched on specific cell types – N-CAM

3. Integrins – major cell-matrix adhesion moleculee.g. 1 integrin, 1 integrin

Cell-Cell adhesion: Cadherin-containing junctions

1. Adherens junction2. Desmosome

-cadherins are a family of calcium-dependentCAMs-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 – continuous band of cadherins found in epithelial cells

-connects cells tightly and interactwith the actin portion of the cytoskeleton

2. desmosomes – also found in high #s inepithelial tissues-cells attach via cadherins – connectto protein “plaques” that attach to theplasma membrane and to intermediatefilaments within the cytoskeleton

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 chains, 8 types of chains• these ab complexes act as receptors to specific matrix components

– e.g. 11 – collagen and laminin– 5 1 - fibronectin

• cells will express multiple integrin types• attachment can be regulated by up- or down-regulated expression of these integrins• 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 junctionsthat 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 betweentwo cells-one important compound small enough to traverse this junction is the second messengercAMP-also allows passage of calcium ions and otherions crucial to signaling (sodium, phosphate)-these gaps are not free-swinging gates

-they actually open and close inresponse to ion concentrations thereby restricting the flow of toomany ions-they also exclude materials basedon size

Cell-Cell and Cell-Matrix interactions can activate signal transduction cascades

• J Biomed Sci. 2006 Feb 23; Crosstalk between hepatocyte growth factor and integrin signaling pathways. Chan PC, Chen SY, Chen CH, Chen HC

• J Neurochem. 2006 Jan;96(1):148-59  Fibronectin promotes brain capillary endothelial cell survival and proliferation through alpha5beta1 and alphavbeta3 integrins via MAP kinase signalling. Wang J, Milner R.

• Immunopharmacol Immunotoxicol. 2005;27(3):371-93.  Interleukin-3, -5, and granulocyte macrophage colony-stimulating factor induce adhesion and chemotaxis of human eosinophils via p38 mitogen-activated protein kinase and nuclear factor kappaB. Ip WK, Wong CK, Wang CB, Tian YP, Lam CW.

• Cell Commun Adhes. 2004 Sep-Dec;11(5-6):137-53. ERK signaling pathways regulate the osteogenic differentiation of human mesenchymal stem cells on collagen I and vitronectin. Salasznyk RM, Klees RF, Hughlock MK, Plopper GE.

• Microsc Microanal. 2005 Jun;11(3):200-8.Cross talk between cell-cell and cell-matrix adhesion signaling pathways during heart organogenesis: implications for cardiac birth defects.Linask KK, Manisastry S, Han M.