SIGNAL TRANSDUCTION SECOND LECTURE

DONE BY: AHMAD ALSAHELE

*ما لون باألسود فهو جزء من الساليدات

*ما لون باألزرق فهو كالم الدكتور

*ما لون باألحمر فهو شرح اضافي

Cell-to-Cell Interactions

-Cells can identify each other by cell surface markers.

-Glycolipids are commonly used as tissue-specific markers

-Major histocompatibility complex (MHC) proteins are used by cells to distinguish "self"

from "non-self"

تم التفصيل عنهما مستقبالً ولهذين البروتينين دور مهم في المناعة وسي MHC-2و MHC-1يوجد منه نوعان MHC*ال

. immunologyفي ال

لخاليا الجسم الواحد ويستطيع الجسم من خاللها تمييز خاصة markersذه البروتينات تعتبر : ه 3*شرح العبارة

األجسام الطبيعية من الغير طبيعية حيث ترتبط هذه البروتينات باألجسام الغير طبيعية وتبرزها للخاليا المناعية ليتم

التخلص منها.

........................................................

Cells within a tissue are connected to each other by cell junctions

1. Tight Junctions – create sheets of cells (they make cells very close together )

2. Anchoring Junctions – connect the cytoskeletons of adjacent cells

3. Communicating Junctions – permit small molecules to pass between cells

a. gap junctions – in animal cells

b. plasmodesmata – in plant cells

........................................................

*the function of

tight junctions is to

make the cells close

together as possible

*anchoring

junctions are

attachment points

for the cytoskeleton

*permit small

molecules to pass

between cells

this projections are called microvilli

they exist in intestinal mucosa

Acetylcholine: common neurotransmitter

بواسطة االنزيم degradationعملية acetylcholine*لما يصير لل

acetylcholinesterase يعود الcholine الى الpresynaptic cell

. reuptake mechanismبعملية تسمى

the common function of

Ach:

opens ligand-gated Na+

channels on muscle cell

and some nerve cells

........................................................

*acetylcholine neurotransmitter act as ligand allows Na+ to pass inner structure of the cell

by change conformation of ligand-gated Na+ channels.

Signal transmitted to muscle cell across a synapse

• the whole mechanism of signal transmission as the

lecturer say in the lecture:

o acetylcholine permit transport of Na+ which

make action potential in postsynaptic

membrane

o action potential move across the axon as

electrical impulse until it reach presynaptic

membrane so that it depolarize

o a.Depolarization opens voltage-gated Ca+2

channels

o b.Ca+2 rushes in; Vesicles fuse with membrane

o c.Neurotransmitter(which is reserved in

presynaptic junction) released; opens ligand-

gated Na+ channels on muscle cell Depolarizes

muscle cell

action potentialوانتهت ب action potential*مما سبق نالحظ أن الرحلة بدأت ب

(action potential=>electrical signal)

Signal: electrical to chemical to electrical

acetate choline

a

c

b

GPCRs that Regulate Ion Channels: Muscarinic Acetylcholine Receptor

*Ach work in striated muscles (nicotinic receptor) & in cardiac muscles (muscarnic

receptors)

The neurotransmitter, acetylcholine (ACH) binds to two types of receptors known as the

nicotinic and muscarinic acetylcholine receptors.

The nicotinic receptor is itself a ligand-gated ion channel that opens on ACH binding. This

receptor is located in the neuromuscular junctions of striated muscle.

بتسمح أليونات Achبس يرتبط فيها ال بوابةعبارة عن nicotinic receptor*في هاي النقطة الزم تعرف انه ال

contractionوفي نهاية المطاف يحصل action potentialومن ثم polarizationالصوديوم انها تدخل وتعمل

للعضلة وتتم الحركة المطلوبة

The muscarinic ACH receptor, is a GPCR found in cardiac muscle cells that is coupled to an

inhibitory G protein

( أي أن سرعة نبض القلب تقل عندما يتم تحفيز inhabitoryمهمة تثبيطية)muscarinic ACH receptor *مهمة ال

. G proteinمستقبالت مرتبطة بهذه المستقبالت التي هي

........................................................

The binding of ACH to this

receptor(muscarinic) triggers dissociation

of Gai-GTP from Gßg, which in this case,

directly binds to and opens a K+ channel.

The movement of K+ down its

concentration gradient to the outside of

the cell, increases [K+] outside which

increases the positive charge outside the

membrane, hyperpolarizing(increase

electrical gradient) the cell. This results in

the slowing of heart rate.

*the main function of Gi protein is : increase K+ concentration out of the cell.

*there are different types of G proteins the most important is: Gi / Gs / G0 / Gq .

*[K+] is more important in heart ,[Na+] is more important in striated muscles.

*if hyperkalemia or hypokalemia happen many problems attack the heart especially in

ECG(a test that checks for problems with the electrical activity of your heart) & intensity of the heart

that can lead to heart attack because of little change in K+ concentration.

*any change(fluctuation) in [K+] may be severe because in the serum and blood the [K+] is

has a small limit(3.5 - 5.5) when we compare with [Na+] (135 - 145)

Structure of GPCRs

G protein-coupled receptors (GPCRs) are the most numerous class of receptors in most

eukaryotes. Receptor activation by ligand binding activates an associated trimeric G

protein, which in turn interacts with downstream signal transduction proteins. All GPCRs

are integral membrane proteins that have a common 7 transmembrane segment

structure (Fig.( The hormone/ligand binding domain is formed by amino acids located on

the external side of the membrane and/or membrane interior (Fig.). GPCRs interact with

G proteins via amino acids in the C3 and C4 cytoplasmic regions.

........................................................

*7 membrane segment protein is

represent 1 receptor only.

*transmembrane coupled receptor

segments are attached to each other by

exterior units(E1,E2,E3,E4) and

cytoplasmic units(C1,C2,C3,C4).

*the most important cytoplasmic units are

C3 C4 because they will interact with G

protein.

G Protein Activation of Effectors

The trimeric G protein cycle of activity in hormone-stimulated GPCR regulation of effector

proteins is summarized in (next slide). Initially, the G protein complex is a chain to the

inner leaflet of the cytoplasmic membrane via lipid anchors attached to the Ga and Gg

subunits. The trimeric GDP-bound form of the G protein is inactive in signaling. The

binding of a hormone to the GPCR triggers a conformational change in the receptor (Step

1) which promotes its binding to the trimeric G protein (Step 2). Binding to the activated

GPCR triggers the dissociation of GDP (Step 3). Subsequent binding of GTP to the Ga

subunit activates it, and causes its dissociation from the receptor and the Gßg complex

(Step 4). Ga-GTP then binds to the effector protein regulating its activity. The hormone

eventually dissociates from the receptor (Step 5). Over time (often less than 1 min), GTP

is hydrolyzed to GDP and Ga becomes inactive. It then dissociates from the effector and

outside

inside

recombines with Gßg (Step 6). A hormone-bound GPCR activates multiple G proteins, until

the hormone dissociates. Proteins known as regulators of G protein signaling (RGS)

accelerate GTP hydrolysis by Ga decreasing the time-period during which Ga is active (not

shown).

........................................................

*ملخص هذه الصورة:

في مما يؤثر receptorبال hormoneيتصل ال

الذي يعد G proteinعلى ال بنيته ويؤثر

(trimeric protein composed of a,b,g)

gو bوهي اكبر من GDPعلى aحيث تحتوي

يحصل receptorبال hormoneعندما يتحد ال

activation ه فيتصل باللG alpha subunit

حول conformational changeويحصل لها

فتتم عملية freeوتصبح GDPال

GTPلتصبح GDPلل phosphrylationال

وتنفصل alpha subunit =< activeفتصبح

أو GDPمرة اخرى GTPيصبح الوعندما activeجاعلة اياه effectorوتتصل مع ال beta & gammaعن

واذا عاد G protein =< trimericيعود كل شيء كما كان وبرجع ال receptorعن ال hormoneينفصل ال

جديدة. cycleتبدأ hormoneال

Trimeric G Proteins & Their Effectors

There are 21 different Ga proteins encoded in the human genome. The G proteins

containing these subunits are activated by different GPCRs and regulate a variety of

different effector proteins . The most common effectors synthesize second messengers

such as cAMP, IP3, DAG, and cGMP. In the case of cAMP, a stimulatory Gs subunit

activates adenylyl cyclase and cAMP production, whereas an inhibitory Gi subunit

inhibits adenylyl cyclase and cAMP production.

*there is another second messengers differ from the mentioned but the mentioned are the

most important.

GPCRs That Bind Epinephrine

Epinephrine is a hormone that signals the "fight-or-flight" response ( اي قاتل او اهرب وفي كال

,It elevates heart rate, cardiac output, blood supply .(الحالتين ستحصل نفس التغييرات للجسم

dilates the airway, and mobilizes carbohydrate and lipid stores of energy in liver and

adipose tissue. In the heart, liver, and adipose tissue, these effects are mediated via

binding to ß1- & ß2-adrenergic GPCRs. Both ß-adrenergic GPCRs signal via Gas, which

activates adenylyl cyclase and raises intracellular [cAMP]. The a2-adrenergic GPCR signals

via Gai, decreasing adenylyl cyclase activity and intracellular [cAMP]. The a1-adrenergic

GPCR is coupled to Gaq, which activates phospholipase C (PLC) and signaling via the

IP3/DAG pathway . a1-adrenergic GPCRs are present in the liver and blood vessels in

peripheral organs. Binding to a1-adrenergic GPCRs stimulates glycogen breakdown in the

liver, while blood flow to peripheral organs is decreased.

glycogenال عن طريق energy productionبسبب زيادة ال peripheral organs*يقل تدفق الدم لل

degredation

GPCRs that Regulate Adenylyl Cyclase

Adenylyl cyclase is an effector enzyme that synthesizes cAMP. Ga-GTP subunits bind to

the catalytic domains of the cyclase, regulating their activity. Gas-GTP activates the

catalytic domains, whereas Gai-GTP inhibits them. A given cell type can express multiple

types of GPCRs that all couple to adenylyl cyclase. The net activity of adenylyl cyclase thus

depends on the combined level of G protein signaling via the multiple GPCRs. In liver,

GPCRs for epinephrine and glucagon both activate the cyclase. In adipose tissue ,

epinephrine, glucagon, and ACTH activate the cyclase via Gas-GTP, while PGE1(or PGE2)

and adenosine inactivate the cyclase via Gai-GTP.

*activation mean increase cAMP while inhibition decrease cAMP

........................................................

حسب احتياجات الخلية. inhibitoryوقد يسيطر تأثير ال stimulatoryال*قد يسيطر تأثير

Activation of Gene Transcription by GPCR Signaling

GPCRs regulate gene transcription by cAMP and PKA signaling. As shown in the figure,

cAMP-released PKA catalytic domains enter the nucleus and phosphorylate the CREB

(CRE-binding) protein, which binds to CRE (cAMP-response element) sequences upstream

of cAMP-regulated genes. Only phosphorylated p-CREB has DNA binding activity. p-CREB

interacts with other TFs to help assemble the RNA Pol II transcription machinery at these

promoters. In liver, glucagon signaling via this pathway activates transcription of genes

needed for gluconeogenesis.

........................................................

*في الصورة التالية يحدث ما يلي:

(denylylلل activationيعمل GPCRفي البداية

(cyclase فيزداد تركيز الcAMP ويتحد الcAMP مع

ثم بصير PKAلل regulatory subunit 2ال

release للcatalytic domins الي بدخلوا

activation(phosphrylation)وبعملوا nucleusال

الي بعمل CREالي يتحد مع ال CREBلبروتين اسمه

activation ومن ثمtranscription .

alzheimerاكتشاف مهم فله عالقة بال CREB*يعد

وله long-term memoryوال Parkinsonوال

وتأثر الجسم drugsعالقة بتأثر الدماغ بال

المختلفة hormonesبال

Down-regulation of GPCR/cAMP/PKA Signaling

A number of events contribute to the termination of signaling by a GPCR. These include:

1. dissociation of the hormone from the receptor,

2. hydrolysis of GTP by Ga

3. hydrolysis of cAMP via cAMP phosphodiesterase,

4. phosphorylation and “desensitization” of receptors by kinases such as PKA and ß-

adrenergic receptor kinase (BARK).

5. In addition, GPCRs can be removed from the membrane by vesicular uptake.

Biological functions mediated by 7TM receptors

1. Smell

2. Taste

3. Vision

4. Neurotransmission

5. Hormone secretion

6. Exocytosis

7. Control of blood pressure

8. Embryogenesis

9. Cell growth and differentiation

10. Development

11. Viral infection

12. Carcinogenesis

G-protein activation

molecular switch

b) Ligand binds

G-protein associates

(c) GDP-GTP exchange

-Subunit dissociates

Active G-Protein-GTP

-> allosteric modulator

of target effector enzyme

*allosteric enzyme : a special type of

enzymes that :-

• have activation site & inhibition site

• have catalytic subunits and regulation subunits

• have 4o structure (it is multisubunit) not only one unit

• are the most important enzymes in metabolism because they had 2 control steps in

metabolism pathways which are responsible of irreversible steps

........................................................

All G-proteins – similar structure/activation

There are TWO broad subclasses of trimeric G-protein-activated signal transduction

pathways:

depends on their target effector enzymes

A. adenylyl cyclase

B. phospholipase C

........................................................

An activated Ga-protein-GTP

– Can trigger the formation of cAMP, which

then acts as a second messenger in

cellular pathways

........................................................

G-protein-GTP activation of Effector

Enzyme adenylyl cyclase produces the

2nd messenger cAMP

Adenylyl Cyclase & Protein Kinase A

Adenylyl cyclase is an integral

membrane protein that contains 12

transmembrane segments (Fig.). It also

has 2 cytoplasmic domains that

together form the catalytic site for

synthesis of cAMP from ATP. One of

the primary targets of cAMP is a

regulatory kinase called protein kinase

A (PKA), or cAMP-dependent protein

kinase.

*الدكتور اكد على هاي األرقام عأساس ما نخلط بينهم:

adenylyl cyclease : 12 transmembrane segments / GPCR : 7 transmembrane segments

trimerc G protein : 3 subunits / PKA : 4 subunits

.segments 6تقسم لمجموعتين كل مجموعة فيها transmembrane segments protein 12*ال

........................................................

PKA exists in two different states inside cells (Fig.). In the absence of cAMP, the enzyme

forms a inactive tetrameric complex in which 2 PKA catalytic subunits are non-covalently

associated with 2 regulatory subunits. When cAMP concentration rises, cAMP binds to

the regulatory subunits which undergo a conformational change, releasing the active

catalytic subunits.

عبارة PKA*الدكتور في هاي الفقرة أكد على انه ال

catalytic & 2 2فيه tetramericعن

regulatory

أما cAMPيتصلوا بالمهمتهم regulatory*ال

activation of next stepمهمتهم catalyticال

PKA =< activeيكون ال cAMP*في حالة وجود ال

inactiveوفي حالة عدم وجوده يكون

........................................................

Protein Kinase A

Phosphorylates downstream target enzymes

activationوظيفتها kinases*ال

وظيفتها phosphrylase*ال

inactivation

What are targets for Protein Kinase A??

cAMP regulated pathways

*the more specific place were water reabsorption take place is distal convolated tubules & collecting ducts.

How to shut it off (the G protein cycle)?

G-protein -subunit is *معنى جملة)

timeron a :)

ما بتكون طول G protien alpha subunitال

في phosphorylationالوقت من بعد عملية ال

بل يوجد هناك وقت محدد تبقى activeحالة ال

ثم تتحول من تلقاء نفسها activeفيه في حالة ال

-auto shutفي عملية تسمى ) inactiveالى

off وقد تصبح هذه العملية اسرع في حال وجود )

GAP =< GTPase activatingبروتين يسمى

protein ( وهذا يفسر عبارةinherent

GTPase activity ولهذا فإنه في حالة عدم )

فإن توقف ligandاستمرار تزويد الخلية بال

بالتالي مسألة وقت ال أكثر و G proteinنشاط ال

وتنتهي استجابة effectorsسيتوقف تحفيز ال

الخلية للهرمون

*auto shut-off => G protein lose its ability to activate target protein that because GTP turn

to GDP and the G protein become inactive

*this process take few seconds or minutes(it is mentioned before in this lecture usually less

than 1 minute)

........................................................

( H2O)بنضيف اله hydrolysisبصيرله cAMP*ال

phosphodiesterase-cAMPبواسطة انزيم

proteinلل activationاول ما يعمل cAMP*ال

kinase وينتهي دوره بصيرلهdegradation وبتحول

ATPثم ADPثم AMPل

: phosphodiesterase-cAMP*هناك مواد تثبط ال

1)caffeine

2)bronchial dilators(such as Theophylline)

وبالتالي بزيد phosphodiesterase-cAMPلما تشرب كافيين يتم تثبيط ال >= caffeine*آلية عمل ال

وهو المطلوب وبهذا فانك ستشعر بالنشاط عند cAMPتاع ال functionأو ال actionال( وبزيد level of cAMP)ال

شرب الكافيين

يستخدم في بعض االمراض التنفسية مثل الربو ويعتبر dilatorbronchialيعتبر Theophylline*ال

drugلهذا ال target proteinهو ال phosphodiesterase-cAMPال

........................................................

phosphorylate <=kinases – phosphatases=> dephosphrylate

Diametrically Opposed…

Remember: whether you active or inactivate by adding P

depends on the specific protein

What if you can’t turn off cascade?

ايش inactiveللبروتين ايش ممكن يصير؟ / لو ما رجع البروتين لحالة ال shut ofحصل ما*معنى السؤال: اذا

ممكن يصير؟ اذا حصل هذا الشيء رح ينصاب الجسم بالعديد من االمراض والمشاكل ومن ضمنها الكوليرا

Vibrio cholera - causes cholera

Normal gut: H20, NaCl, NaHCO3 secretion controlled by hormones via Gs/cAMP signal

pathways

بشكل منظم وكميات معقولة intestineفي الوضع الطبيعي األيونات تفرز الى ال*

V. cholera – secretes enterotoxin, chemically modifies Gs – no GTPase activity - stays ON

تساهم في جعل عملية االفراز مستمرة )ببطل في عملية toxic proteins*في الكوليرا تفرز البكتيريا المسببة للمرض

inhibition للcAMP وبضل البروتينactive وبصير في خلل بالmembrane )

Severe watery diarrhea – dehydration, death

,مما يؤدي الى الجفاف وبالتالي الموت diarrheaمرض نتيجة ال*يتم فقدان الكثير من السوائل بسبب هذا ال

*يموت سنوياً مئات اآلالف من دولة اليمن الشقيقة نتيجة الكوليرا الناتجة عن التلوث وبخاصة تلوث المياة

target effector enzyme is Phospholipase C

PLC cleaves a membrane phospholipid (Phoshatidyl

inositol) to

two 2nd Messengers:

Inositol-1,4,5-Trisphosphate(InsP3) &

Diacylglycerol (DAG)

........................................................

*you should know :

1. DAG

a. 2 fatty acids + glycerol

b. lipid soluble

2. InsP3

a. 3 phosphate groups +suger nucleus

b. water soluble

GPCRs That Activate Phospholipase C

Another common GPCR signaling pathway involves the activation of phospholipase C

(PLC). This enzyme cleaves the membrane lipid, phosphatidylinositol 4,5-bisphosphate

(PIP2) to the second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol

(DAG) (Fig.). In this case, the Go and Gq G proteins conduct the signal from the GPCR to

PLC. This is the pathway used in a1-adrenergic GPCR signaling in the liver.

,phosphatidylethanolamineهما نوعين: phospholipidة لتشابتر سابق أهم انواع ال*مراجع

phosphatidylcholine

IP3/DAG Signaling Elevates Cytosolic Ca2+

*Ca++ is important for neurotransmitters

The steps downstream of PLC that make up the IP3/DAG signaling pathway are illustrated

in the figure. IP3 diffuses from the cytoplasmic membrane to the ER where it binds to and

triggers the opening of IP3-gated Ca2+ channels (Steps 3 & 4). Another kinase, protein

kinase C (PKC) binds to DAG in the cytoplasmic membrane and is activated (Step 6). In

liver, the rise in cytoplasmic [Ca2+] activates enzymes such as glycogen phosphorylase

kinase, which phosphorylates and activates glycogen phosphorylase. Glycogen

phosphorylase kinase is activated by Ca2+-calmodulin. In addition, PKC phosphorylates

and inactivates glycogen synthase.

........................................................

*IP3

increase [Ca++] in cytosol from ER

Ca++ ==> activate PKC

PKC ==> do its function

*DAG

activate PKC

PKC ==> phosphorylation of substrates

........................................................

DAG

Activates

Protein

Kinase C

(Starts

Cascade)

..........

InsP3

Ligand

for ER

ligand-

gated

Ca++

channels

when ligand-gated Ca++ channels open the cell response occur

*the form at which Ca++ present in the cell is calcium-calmodulin complex

........................................................

Response:

Protein Kinase C phosphorylates target proteins (ser & thr)

cell growth

regulation of ion channels

cytoskeleton

increases cell pH

Protein secretion

Ca++

Binds & activates calmodulin Calmodulin-binding proteins activated (kinases &

phosphatases)

Signal Trans. Components: GTPase Switches

GTPase switch protein also play important roles in intracellular signal transduction .

GTPases are active when bound to GTP and inactive when bound to GDP. The timeframe

of activation depends on the GTPase activity (the timer function) of these proteins.

Proteins known as guanine nucleotide-exchange factors (GEFs) promote exchange of GTP

for GDP and activate GTPases. Proteins known as GTPase-activating proteins (GAPs),

stimulate the rate of GTP hydrolysis to GDP and inactivate GTPases.

GTPase-عكس ال responseوظيفته يسرع ال GEFs( exchange factors-guanine nucleotide(*البروتين

)GAPs( activating proteins الي وظيفته يثبط

........................................................

We will cover two classes of GTPase switch proteins--trimeric (large) G proteins, and

monomeric (small) G proteins. Trimeric G proteins interact directly with receptors,

whereas small G proteins interact with receptors via adaptor proteins and GEFs.

activating proteins-GTPaseال *

accelerating-GTPase الهوه نفسه

proteins

Signal Trans. Components: 2nd Messengers

While there are a large number of extracellular receptor ligands ("first messengers"),

there are relatively few small molecules used in intracellular signal transduction ("second

messengers"). In fact, only 6 second messengers occur in animal cells. These are cAMP,

cGMP, 1,2-diacylglycerol (DAG), and inositol 1,4,5-trisphosphate (IP3) , and calcium and

phosphoinositides . Second messengers are small molecules that diffuse rapidly through

the cytoplasm to their protein targets. Another advantage of second messengers is that

they facilitate amplification of an extracellular signal.

........................................................

*the most common 2nd messengers

Nitric Oxide (NO)/cGMP Signaling

A related signaling pathway involving phospholipase C operates in vascular endothelial

cells and causes adjacent smooth muscle cells to relax in response to circulating

acetylcholine (Fig.) In the NO/cGMP signaling pathway, the downstream target of

Ca2+/calmodulin is nitric oxide synthase, which synthesizes the gas NO from arginine. NO

diffuses into smooth muscle cells and causes relaxation by activating guanylyl cyclase and

increasing [cGMP]. As a result arteries in tissues such as the heart dilate, increasing blood

supply to the tissue. NO also is produced from the drug nitroglycerin which is given to

heart attack patients and patients being treated for angina.

حيث انه يعمل على زيادة تركيز nitroglycerin دواء ال(angina*يتم اعطاء األشخاص المصابين بالذبحة الصدرية)

ويعالج المصاب بالذبحة الصدرية vasodilationالذي يحدث NOال

........................................................

"second messenger"

هو تحفيز 2nd messengers*الهدف من كل هذه ال

لتقوم target proteinsيحفز اللكي PKCال

. responseبال

Metabolism of Diacylglycerol

Diacylglycerol may be (1) phosphorylated to phosphatidate or (2) hydrolyzed to glycerol and fatty acids(degraded).

Summary

{{{ THE END }}}