mechanism of action-2 › presentation › c4c3 › 12dfeb60637… · mechanism of action-2 05 •...

• receptor turnover, something to keep in mind

• table: hormone receptors and their effectors

• from hormones to their actions, 7tmr and single tmr, amplification, crosstalk, diseases

• signaling through G-protein linked receptor linked to the enzyme AC or the enzyme PLC (seven transmembrane domain receptors)

• signaling through receptors having or not having intrinsic tyrosine kinase activity, the mitogenic and the cytokine activated pathways (single transmembrane domain receptors)

• signaling through nitric oxide and receptors linked to the enzyme Guanylate Cyclase

• sequence of endocrine events involved in the activation of endocrine glands and their targets

Mechanism of Action-2

05

• THE STORY LINE

• signal to system

• endocrine gland

• hormone secretion

• H transport

• H - R interaction

• target response

Today’s lecture

Today’s lecture

Today’s lecture

S E

sensor

integrator

center

effector

afferent

“story line” efferent

“story line”

negative feedback

“story line”

diagram for a control system

as that present in a refrigerator

a “reflex arc”

a base for a

control model

S

E … if story lines are linked through an

integrator, then you have “control”…

Today’s lecture

“story

line”

I suggest you put this information into a table YOU design !!!

Membrane Hormone Receptors

How receptors get to the membrane and what might happen after they bind its ligand


Adenylate cyclase

ACTH, ßadrenergic Cas(Gs),LH, HCG, FSH, Glucagon, PGs, PTH,

TSH, alpha adrenergic (Gi), SS

Guanylate cyclase

ANP (ANF)

Phoshoinositol turnover

and Ca flux

Ach receptor (muscarinic), alpha adrenergic Cas, Ang II

(also activates Tyr-kinase),

GnRH (LHRH), TRH, AVP

Receptor protein

tyrosine kinase

(mitogenic pathway)

Insulin, IGF, EGF, CSF, FGF

Receptor - associated tyrosine kinase

(cytokine pathway)

GH, PRL, EPO, Interleukins,

NGF, T-cell receptors

Ion channels

Ach receptor (nicotinic), GABA

Membrane receptors and their ligands

GPCR to AC

GPCR to PLC

7tmr

single tmr


plasma

memb.

COOH

2 ECF

ICF

seven - transmembrane

domain receptors

ß - adrenergic and

glucagon receptors among many others

EGF insulin

PDGF ANP

GH,

Prl, cytokines

kinase

Cys rich

Cys residues

JAK2

ECF

ICF

COOH

N H

N H2

hydrophobic aa

4 - tm domain receptors

are usually ion channels

single - tm

domain receptors

G



GPCR

to AC

An unactivated G-protein coupled receptor. The seven transmembrane alpha helices are connected by three extracellular and three intracellular loops of varying length. The extracellular loops may be glycosylated, and the intracellular loops and C- terminal tail may be phosphorylated. The receptor is coupled to a G-protein consisting of a GDP-binding -subunit bound to a / component. The and subunits are tethered to the membrane by lipid groups.

Note: next 9 slides

tell this same story

G-protein coupled receptors (or 7 transmembrane domain)

GPCR

7tmr

GPCR

to AC

H

R

C ECF

ICF

H

R

R ECF

ICF Gs

GDP

replaced by GTP

GTP

cAMP

H

R

C ECF

ICF GDP

GTPase activity of the subunit is blocked by Cholera toxin

ACTIVE INACTIVE

AC

PK

Gs

Gs

GPCR

to AC



cAMP

GPCR

to AC

Activation of G-protein coupled receptor. (I) Resting state. (II) Hormone binding produces a conformational change in the receptor that causes (III) the subunit to exchange ADP for GTP, dissociate from the / -subunit and interact with its effector molecule. The / -subunit also interacts with its effector molecule. (IV) The subunit converts GTP to GDP, which allows it to reassociate with the / -subunit, and the hormone dissociates from the receptor, restoring the resting state. (I). GTPase activity of the alpha-subunit is blocked by Cholera toxin.

Formation and degradation of

cyclic adenosine monophosphate

ATP

AMP

Pyrophosphate


cAMP

GPCR

to AC

Effects of cyclic AMP.I. Activation of protein kinase A accounts for most of the cellular actions of cyclic AMP. Inactive protein kinase consists of two catalytic units (C), each of which is bound to a dimer of regulatory units (R). When two molecules of cyclic AMP bind to each regulatory unit, active catalytic subunits are released. Phosphorylation of enzymes, ion channels, and transcription factors of the CREB (cyclic AMP response element binding) family activates or inactivates these proteins. II. Cyclic AMP also binds to the -subunits of cyclic nucleotide-gated ions channels (lower portion of the figure) causing them to open and allow influx of sodium and calcium. III. Cyclic AMP binds to and activates the nuclear exchange factors (EPAC: exchange proteins activated by cyclic AMP), which in turn activate the small G-protein RAP-1.


GTPase activity of the subunit is blocked by Cholera toxin

GPCR

to AC

CREB

is a TF

CRE

GPCR

to AC

Amplification at the subcellular level: a

cascade effect


Push - Pull at the subcellular level: a control effect

GPCR

to AC


GPCR

to AC



GPCR

to PLC

Phosphatidylinositol-bisphosphate gives rise to inositol 1,4,5 trisphosphate (IP3) and diacylglycerol (DAG) when cleaved by phospholipase C. R1 and R2 = long chain fatty acids. The numbered angles in the hexagon represent carbon atoms of inositol.

Diacylglycerol (DAG). Formed from phosphatidyl inositol 4,5 bisphosphate by the action of phospholipase C, may be cleaved by DAG lipase to release arachidonate, the precursor of the prostaglandins and leukotrienes.


GPCR

to PLC

GPCR

7tmr


GPCR

to PLC

• Epi, NE, Ach, AVP, Ang II, 5HT, SP, DA, LH, FSH, TSH, PAF, PG, glucagon, rhodopsin

• Gs (AC), Gi (AC), Gq (PLC) are hetero-trimers ( , , ). The subunit has GTPase activity

• through G protein, receptor is linked to either an enzyme or a channel to express the effect of the bound ligand

• dissociation of GDP and binding of GTP to the alpha subunit is elicited by ligand binding to the receptor

• the Gs family couples to an increase Adenylate Cyclase (AC) activity and opens K channels

• the Gi family couples to and decrease AC activity, open K and closes Ca channels

• the Gq family activates Phospho Lipase C (PLC) to increase Inositol - 3P (IP3), Di - Acyl Glycerol (DAG), and intracellular Ca

H

R enzyme

ECF

ICF GDP

replaced by GTP

Gs

H

R channel

ECF

ICF GDP

replaced by GTP

Gs

Overview on plasmalema GPCR

• Nobel prize for cAMP and G proteins

• Rs / Gs: Epi, ACTH, AVP, Ang II, LH, FSH, TSH, 5HT, glucagon

• Ri / Gi: 2 adrenergic, muscarinic - Ach agonists, opioids

• cAMP regulates cell functions by binding to cAMP - dependent PKA

• FSH on follicles, Ca channels, CREB

• glycogen phosphorylase (+) vs. synthetase (-), hormone sens. lipase (+)

• AVP / V2 / Aq3, cystic fibrosis transmembrane regulator (CFTR, Cl channel)

• ANP effects (vasodilatation, natriuresis, diuresis) mediated by production of cGMP probably maintain cation channels open, thus explaining its natriuretic action


• membrane actions of most hormones involve generation of intracellular cyclic nucleotides as second messengers and stimulation of PKs.

• cyclic nucleotide - independent substrate phosphorylation may be important in the action of hormones such as insulin and growth factors.

• In addition to protein kinases (PKs), protein phosphatases might be also important targets for cellular regulation.

• signal transduction associated cascade effects as an amplification mechanism for hormone signals

• cross-talk mechanisms as an interaction / integration mechanism for hormone signals (e.g. --------------------->)



Agonist removal

Receptor uncoupling

Receptor endocytosis

Receptor downregulation

(ending GPCR action)

• Ach / muscarinic, Epi / NE / alpha 1, AVP / V1 (liver)

• Gq/ PLC/ IP3/ DAG

• the IP3 receptor is a Ca channel

• Ca - calmodulin complex activates enzymes such as phosphorylase kinase and cAMP phosphodiesterase

• DAG activates PKC

• PKC also (+) AP-1

• Some hormones, often referred as first messengers, interact with cell membrane to increase production of 2nd messengers (e.g.. cAMP, cGMP) more directly responsible for activation of the cell. Cyclic nucleotides mediate their actions through cellular kinases and specific phosphorylated proteins. However, the 1st / 2nd messenger model must now be expanded to include other messengers in its temporal sequence. For example ….

• …. some hormones require Ca for activation of cyclic nucleotide formation. In some cells the hormone acting through an inward Ca flux, may activate PG biosynthesis which activates cAMP or cGMP.


single tmr


Phosphorylation of tyrosines on dimerized receptors (R) following hormone (H) binding provides docking sites for the attachment of proteins that transduce the hormonal signal. The growth factor binding protein 2 (GRB2) binds to a phosphorylated tyrosine in the receptor, and binds at its other end to the nucleotide exchange factor SOS, which stimulates the small G-Protein Ras to exchange its GDP for GTP. Thus activated, Ras in turn activates the protein kinase Raf, which phosphorylates mitogen activated protein (MAP) kinase and initiates the MAP kinase cascade that ultimately phosphorylates nuclear transcription factors. The isoform of phospholipase C (PLC ) docks on the phosphorylated receptor and is then tyrosine phosphorylated and activated to cleave phosphatidyl inositol 4,5 bisphosphate (PIP2) releasing diacylglycerol (DAG) and inositol tris phosphate (IP3) and activating protein kinase C (PKC).

GPCR to AC

GPCR to PLC

7tmr

single tmr



Adenylate cyclase

ACTH, ßadrenergic Cas(Gs),LH, HCG, FSH, Glucagon, PGs, PTH,

TSH, alpha adrenergic (Gi), SS

Guanylate cyclase

ANP (ANF)

Phoshoinositol

turnover and Ca flux

Ach receptor (muscarinic), alpha adrenergic Cas, Ang II

(also activates Tyr-kinase),

GnRH (LHRH), TRH, AVP

Receptor protein

tyrosine kinase

(mitogenic pathway)

Insulin, IGF, EGF, CSF, FGF

Receptor - associated tyrosine kinase

(cytokine pathway)

GH, PRL, EPO, Interleukins,

NGF, T-cell receptors

Ion channels

Ach receptor (nicotinic), GABA

Membrane receptors and their ligands

EGF

insulin

PDGF ANP

GH

Prl cytokines

kinase

Cys

rich Cys residues

JAK2

ECF

ICF

• These receptors contain an intrinsic protein kinase that catalyzes phospho-rylation of tyrosine residues (mitogenic path) or “borrow” a JAK kinase to do it (cytokine path)

• Cystein and its thiol (SH) group linking the aa to another Cystein form part of a binding cleft for ligands

NH2

COOH

Receptors with intrinsic Tyr-kinase activity (single transmembrane)

membrane hydro-

phobic aa

Cytokine activated pathway

• These receptors contain an intrinsic protein kinase that catalyzes phospho-rylation of tyrosine residues (mitogenic path) or “borrow” a JAK kinase to do it (cytokine path)

• Cystein and its thiol (SH) group linking the aa to another Cystein form part of a binding cleft for ligands



Dimerized hormone receptors (R) associate with the JAK family of cytosolic protein tyrosine kinases, and become phosphorylated on tyrosines. Proteins of the STAT family (S) of transcription factors that reside in the cytosol in the unstimulated state are recruited to the phosphorylated receptor. After phosphorylation by JAK, STATs dissociate from the receptor, form homodimers, and migrate to the nucleus where they activate gene transcription.

• receptors for cytokines (IL), GH, and Prl, do not have a kinase domain

• dimerization, if GH excess inhibition

• ligand binding results in rapid phosphorylation of cell proteins on Tyr

• Janus (JAK) kinases are cytoplasmic tyrosine kinases which physically associate with the box 1 - box 2 domains of the ligand bound receptor leading to auto - phosphorylation on Tyr residues and phosphorylation of transcription factors called “signal transducers and activators of transcription” or in short “STATS”

JAK2 STAT

DNA

GH





EGF

insulin

PDGF ANP

GH

Prl cytokines

JAK2

ECF

ICF

NH2

COOH

membrane

Mitogen activated pathway

• These receptors contain an intrinsic protein kinase that specifically catalyzes the phosphorylation of tyrosine residues on proteins

• Cystein and its thiol (SH) group linking the aa to another Cystein aa may form part of a binding cleft for ligands


EGF insulin

(the previous pathway)


PDGF








(eg)



(eg)

WIKIPEDIA:

Akt/PKB is a

serine/threonine

protein kinase

that plays a key

role in multiple

cellular processes

such as glucose

metabolism, cell

proliferation,

apoptosis,

transcription and

cell migration.

Family members In humans, there are three genes in the "Akt family":


WIKIPEDIA:

Akt/PKB is a

serine/threonine

protein kinase

that plays a key

role in multiple

cellular processes

such as glucose

metabolism, cell

proliferation,

apoptosis,

transcription and

cell migration.


(e.g. AMH)

GPCR to AC

GPCR to PLC

7tmr

single tmr


Gases as neural messengers glutamate

NMDA

receptor

Ca

calmodulin

+

NOS inactive NOS active

calmodulin

+ Ca

ion channel

arginine

citrulline

NO

NO - Fe

guanylyl cyclase

GTP

cGMP

physiological

response

• NO formed by conversion of arginine to citrulline by the enzyme NO synthase. NO plays a role in neuropeptide release controlling ant. pituitary (GnRH and CRH)

• NO binds to a soluble GC to stimulate the formation of cGMP which in turn activates ion channels and PKG

Prostaglandins and Leukotrienes

membrane phospholipids

arachedonic acid

PGG2 5HPTE

leukotrienes prostacycline

PGI2

prostaglandins

PGE2 PGF2a

PGD2

tromboxane A2

tromboxane B2

PLA2

cyclooxygenase

(blocked by NSAID) lipooxygenase

prostacycline

synthetase

prostaglandin

synthetase

tromboxane

synthetase

Crosstalk at the subcellular level: an integration mechanism

Sequence of endocrine events involved in the activation of endocrine

glands and their targets

• signal to endocrine and neuroendocrine systems

• endocrine gland and their receptors to their inputs

• hormone secretion in response to input signals

• hormone transport in blood, degradation, free vs bound

• hormone - receptor interaction and signal transduction

• target responses by genomic vs non-genomic pathways

• evolution and signal transduction pathways

mechanism of action-2 › presentation › c4c3 › 12dfeb60637… · mechanism of action-2 05 •...

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