Cell signaling

INTRODUCTION TO ENDOCRINE SIGNALING

GENERAL OUTLINE OF CELL SIGNALING AND SIGNAL TRANSDUCTION

SIGNALING MOLECULES AND RECEPTORS

S I G N A L T R A N S D U C T I O N A N D S I G N A L M E D I AT O R S

MECHANISMS OF ACTIONS OF SIGNALING MOLECULES

Learning outcomes
Understand the general characteristics and properties of signaling molecules and signal transduction process

Outline the process of cell signaling and signal transduction

Define receptors and describe the different types of receptors involved in the cell signaling process

Describe the mechanism of action of the lipophilic signaling molecules

Describe the mechanism of action of signaling via G-protein coupled receptors

Explain the role of second messengers in the signal transduction process

Describe the mechanism of action of signaling via enzyme-linked receptors, specifically receptor guanylyl cyclase,
receptor tyrosine kinase, and tyrosine kinase-associated receptor

Understand the biochemical basis of endocrine disorders and the basis of endocrine function assessment

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Outline of lecture contents
Introduction to endocrine signaling Mechanisms of actions of signaling molecules
◦ Hormones – chemical nature, biosynthesis, modification,
Mechanism of action of lipophilic signaling molecules
transportation

◦ Hormonal interaction Mechanism of action of signaling molecules via ion-

channel linked receptor
◦ Control of hormone secretion and plasma hormone level

◦ Biochemical basis of endocrine disorders and endocrine Mechanism of action of signaling molecules via GPCR

function assessment • cAMP signaling cascade

• Phosphoinositide signaling cascade

General outline of cell signaling and signal transduction
Mechanism of action of signaling molecules via enzyme
Signaling molecules and receptors linked receptor
• Signaling via receptor guanylyl cyclase/cGMP
Signal transduction and signal mediators
• Signaling via receptor tyrosine kinase

• Signaling via tyrosine kinase associated receptor

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Introduction to endocrine signaling
Intercellular communication and signaling is essential for body homeostasis mechanisms, adaptation
to constantly changing environment, cell growth, division and differentiation

Two major systems of intercellular communication

◦ Nervous system

◦ Endocrine system

Endocrine system
◦ Mediated through hormones which serve as mobile messages

◦ Systemic or general hormones – act on distant targets (via blood stream), act on various target tissues, thus;
generating widespread effects

◦ Local hormones – act locally (mainly in the tissues and sites in which they are produced)

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Hormones
A chemical messenger, secreted
in trace amount by specific cells
that carries a signal to generate
some alteration at the cellular
level

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J. Koolman and K-H. Roehm, Color Atlas of Biochemistry, 2nd edition, pp. 371; © 2005 Thieme.
 Juxtacrine signaling Paracrine signaling Synaptic signaling

Forms of
intercellular
signaling

Endocrine signaling Autocrine signaling

Alberts et.al., Molecular biology of the cell, 6th edition, pp. 815; © 2015 by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, and Peter Walter.
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M. Lieberman, A.D. Marks and A. Peet, Marks' Basic Medical Biochemistry: A clinical approach, 4th edition, pp. 175; © 2013 Lippincott Williams & Wilkins, a Wolters Kluwer business.
Structurally and chemically diverse nature of hormones Major endocrine organs in the body
R.L. Miesfeld and M.M. McEvoy, Biochemistry, 1st edition, pp. 377; © 2017 by W. W. Norton & Company, Inc.
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D.L. Nelson, M.M. Cox, Lehninger Principles of Biochemistry, 6th edition, pp. 936; ©2013, 2008, 2005, 2000 by W. H. Freeman and Company.
J.W. Baynes and M.H. Dominiczak, Medical biochemistry, 5th edition, pp. 370; © 2019, Elsevier Limited.

V.W. Rodwell et al., Harper's illustrated biochemistry, 30th edition, pp. 502; © 2015 by The McGraw-Hill Education.

The chemical nature of hormones

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 Hormones can be chemically diverse in nature

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D.L. Nelson, M.M. Cox, Lehninger Principles of Biochemistry, 6th edition, pp. 933; ©2013, 2008, 2005, 2000 by W. H. Freeman and Company.
 Biosynthesis and modification of hormones

Synthesized and secreted

• e.g., estriol, aldosterone, cortisol, catecholamines
as “active forms”

Synthesized as
• e.g., insulin, parathyroid hormone (PTH)
“preprohormones” and
• ACTH, β-lipotrophin, γ-MSH derived from cleavage of POMC (pro-opiomelanocortin) peptide
secreted as “active forms”

• Conversion of active form in target tissues e.g., T3 to T4 conversion, conversion of testosterone to dihydrotestosterone (DHT)
Synthesized and secreted
• Conversion of active form in non-target tissues e.g., conversion of DHEA to androstenedione and testosterone in liver
as “prohormones” • Conversion of active form requires both target and non-target tissues e.g., 1, 25 DHCC formation

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 Biosynthesis and modification of hormones

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D.L. Nelson, M.M. Cox, Lehninger Principles of Biochemistry, 6th edition, pp. 934; ©2013, 2008, 2005, 2000 by W. H. Freeman and Company.
Transportation of hormones
Transported as free form or bound form in circulation

Water soluble hormones – not require transport proteins, transported as free forms

Lipid soluble hormones – require specific carrier proteins, transported as bound forms
◦ Glucocorticoids → corticosteroid binding globulin (CBG) or transcortin

◦ Sex hormones → sex hormone binding globulin (SHBG) or testosterone-estrogen binding globulin
(TEBG)

Only unbound or free form – biologically active

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Hormonal interaction

Opposite effects on
target tissues
Inhibitory Interaction
e.g., insulin and
glucagon

Produce effects greater e.g., FSH and LH on

than the sum of either follicular growth,
of them giving alone or
Synergistic Interaction GH, T3 and insulin
producing new effect on optimal growth

Presence of a small quantity e.g., corticosteroid

of one hormone allows full facilitates the pressor
Permissive Interaction effects of
response of another
hormone catecholamines

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 Control of hormone secretion and plasma hormone level

Major determinant factors of plasma hormone level

◦ Glandular secretion pattern (rate of secretion)

◦ Hormone clearance rate

Regulation of hormone secretion

◦ Negative feedback regulation upon increased plasma
hormone level

◦ Positive feedback regulation upon decreased circulatory

hormone level

Some hormone secretion is regulated via a regulatory

axis e.g., hypothalomo-pituitary-target endocrine
organ axis

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J.W. Baynes and M.H. Dominiczak, Medical biochemistry, 5th edition, pp. 370; © 2019, Elsevier Limited.
Neuro-
endocrine
axis

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D.L. Nelson, M.M. Cox, Lehninger Principles of Biochemistry, 6th edition, pp. 937; ©2013, 2008, 2005, 2000 by W. H. Freeman and Company.
D.L. Nelson, M.M. Cox, Lehninger Principles of Biochemistry, 6th edition, pp. 930; ©2013, 2008, 2005, 2000 by W. H. Freeman and Company.
T. Devlin Ed., Textbook of Biochemistry with clinical correlations, 7th edition, pp. 886; © 2011, 2006, 2002, 1997, 1992, 1986, 1982 John Wiley & Sons, Inc.

Neuro-endocrine axis

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endocrine organ
Hypothalamo-
pituitary-target

axis
Endocrine disorders Biochemical basis of endocrine disorders
Two categories:

• Hormonal excess Hormone excess Hormone deficiency

• Hormonal deficiency
Overactive Defects in
Hormones Hormones
hormonal hormonal
overproduction underproduction
signaling pathway signaling pathway

Overactive or Autoimmune Altered tissues

Autoimmune cause
If pathology lies in overexpression of destruction e.g., response to
Hypothalamus signal mediators
e.g., Grave’s disease
type 1 DM hormones
this level → TERTIARY

If pathology lies e.g., activation Mutation of

Pituitary in this level → mutation of LH hormone receptors
Endocrine neoplasia Surgical removal
SECONDARY receptor in e.g., Laron’s
precocious puberty dwarfism

Target endocrine If pathology lies

in this level →
Infection e.g., TB
organ Exogenous causes infection of adrenal Insulin resistance
PRIMARY
gland
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Biochemical assessment of endocrine function
To determine whether the
specific endocrine system is
functioning abnormally Peripheral hormone receptors level

To localize the functional defect

Measurement of hormone binding
Assessment of at least two
proteins
points in endocrine feedback
Endocrine function assessment
loop axis Evoked or suppressed hormones level
assessment

Determination of basal circulating

hormone level

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Signal transduction
Process by which the message carried by signaling molecule is accepted by the specific receptor and is transmitted via
intracellular signal mediators to generate the appropriate responses within the cells
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J.M. Berg, J.L. Tymoczko, G.J. Gatto, Jr., L. Stryer, Biochemistry, 8th edition, pp. 398; ©2015, 2012, 2007, 2002 by W. H. Freeman and Company; © 1995, 1988, 1981, 1975 by Lubert Stryer.
General outline of cell signaling or signal Changes in external and internal environment (Stimulus)
transduction Synthesis and secretion of signaling molecules in
Release of primary messenger
response to stimulus

 Blood glucose level during fed state

Reception of primary messenger or signaling molecules

Increased insulin secretion from Binding of signaling molecules to the receptor on Initiates transfer of information from signaling
target cells molecules into the cells
pancreatic β cells

Binding to insulin to insulin receptors Delivery of message inside the cell by second messengers/intracellular signal mediators
on target cells
Involve signal mediators such as G protein, second
Activation of intracellular signaling pathways
messengers, protein kinases, DNA binding proteins

 Glucose uptake and utilization in

target cells
Activation of effectors that directly alter the physiological response
Changes in cellular metabolism, or gene expression
Alter the activity of effector proteins
or behavior
Blood glucose level return to normal

Termination of the signal

Reduce insulin secretion from pancreas
Removal of signaling molecules or receptors or inactivation of intracellular signaling events
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General outline of cell signaling

D.L. Nelson, M.M. Cox, Lehninger Principles of Biochemistry, 6th edition, pp. 939; ©2013, 2008, 2005, 2000 by W. H. Freeman and Company.
or signal transduction

• Release of primary messenger

in response to stimulus

• Reception of primary
messenger

• Delivery of message inside the

cell

• Activation of effectors that

directly alter the physiological
response

• Termination of signal

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R.L. Miesfeld and M.M. McEvoy, Biochemistry, 1st edition, pp. 373; © 2017 by W. W. Norton & Company, Inc.
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Alberts et.al., Molecular biology of the cell, 6th edition, pp. 814; © 2015 by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, and Peter Walter.
Changes in the cells in response to hormone action may occur
immediately (fast action) or at a slow rate (slow action); may
produce short-term or long-term effects depending on the effects
on target proteins

Alberts et.al., Molecular biology of the cell, 6th edition, pp. 826; © 2015 by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, and Peter Walter.
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D. Sadava et al., Life: The Science of Biology, 10th edition, pp. 127; © 2014 by Sinauer Associates, Inc.
 Features of signal
transduction
systems

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D.L. Nelson, M.M. Cox, Lehninger Principles of Biochemistry, 6th edition, pp. 434; ©2013, 2008, 2005, 2000 by W. H. Freeman and Company.
Signaling molecules

D.L. Nelson, M.M. Cox, Lehninger Principles of Biochemistry, 6th edition, pp. 436; ©2013, 2008, 2005, 2000 by W. H. Freeman and Company.
Molecules that can generate specific
response in its target cell to adapt the
changes in its environment

• Hormones (major factor)

• Growth factors and cytokines

• Neurotransmitters

• Cellular antigens and extracellular

matrix components

• Special senses (light, sound, taste,

smell, touch)

• Gases (nitric oxide, free radicals)

• Other physicochemical factors (pH,

pO2, temperature, osmolarity, etc.)

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R.L. Miesfeld and M.M. McEvoy, Biochemistry, 1st edition, pp. 377; © 2017 by W. W. Norton & Company, Inc.

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 A given hormone can affect several different cell types that express its
Target cell concept receptors

Hormones can only act on the cells that express its receptors (called TARGET CELLS of that hormone)

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L.A. Urry et al., Campbell Biology, 11th edition, pp. 228; © 2017, 2014, 2011 Pearson Education, Inc.
 Receptors
Proteins or glycoproteins in nature

Contains two functional domains

• Signaling recognition/binding
domain

• Signal transduction domain

Types of receptors

• Intracellular receptors

• Cell surface or cell membrane

receptors
Cell associated recognition proteins that are able to recognize and bind specific
extracellular signaling molecule (ligand) and initiate intracellular signaling
pathway to generate the appropriate response in the cell
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M. Lieberman, A.D. Marks and A. Peet, Marks' Basic Medical Biochemistry: A clinical approach, 4th edition, pp. 171, 176; © 2013 Lippincott Williams & Wilkins, a Wolters Kluwer business.
 SPECIFICITY OF CELL SIGNALING

Hormone receptors show high specificity and high affinity to its hormones

L.A. Urry et al., Campbell Biology, 11th edition, pp. 228; © 2017, 2014, 2011 Pearson Education, Inc.
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V.W. Rodwell et al., Harper's illustrated biochemistry, 30th edition, pp. 498; © 2015 by The McGraw-Hill Education.
 Intracellular receptors
Cytosolic or nuclear receptors

Act as gene specific transcription factors

Receptor itself acts as intracellular signal

mediator

Functional domains of intracellular

receptors

• Hormone binding domain

• DNA binding domain

• Transcription regulatory domain

• Cofactor binding domain

Receptors for lipophilic signaling molecules such as steroid/thyroid hormones
• Translocation domain
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R.L. Miesfeld and M.M. McEvoy, Biochemistry, 1st edition, pp. 417; © 2017 by W. W. Norton & Company, Inc.
General signaling mechanism
mediated by intracellular receptors

• Act as gene-specific
transcription factors

• Receptor itself carry signal,

thus, act as intracellular signal
mediators

• The hormone-receptor
complex binds to specific DNA
region and influence the rate of
target gene transcription

D.L. Nelson, M.M. Cox, Lehninger Principles of Biochemistry, 6th edition, pp. 472; ©2013, 31
2008, 2005, 2000 by W. H. Freeman and Company.
 Cell surface or cell membrane receptors
T. Devlin Ed., Textbook of Biochemistry with clinical correlations, 7th edition, pp. 508; © 2011, 2006, 2002, 1997, 1992, 1986, 1982 John Wiley & Sons, Inc.

Receptors for hydrophilic signaling

Classified based on types of mechanism
molecules
used for signal transduction
Act as signal transducers (cannot act as

Ion-channel linked receptors intracellular signal mediator)

Convert extracellular signal into one or

G-protein coupled receptors (GPCRs) more intracellular signals

Three functional domains

Enzyme linked receptors
• Hormone binding domain

• Signal transduction or catalytic

domain

• Transmembrane domain

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 Ion channel linked receptor
D.L. Nelson, M.M. Cox, Lehninger Principles of Biochemistry, 6th edition, pp. 466; ©2013, 2008, 2005, 2000 by W. H. Freeman and Company.

• Transmitter gated ion channels

• Homologous multi-pass
transmembrane proteins

• Involved in rapid synaptic signaling

between electrically excitable cells

M. Lieberman, A.D. Marks and A. Peet, Marks' Basic Medical Biochemistry: A clinical approach,
4th edition, pp. 173-4; © 2013 Lippincott Williams & Wilkins, a Wolters Kluwer business.
• Transient opening or closing of ion
channels associated with receptors
upon binding with
neurotransmitters

• Mediate synaptic signaling by

changing the ion permeability and
excitability of the post-synaptic
cells e.g., acetylcholine receptors

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G protein coupled receptors (GPCRs)
Homologous seven-pass transmembrane proteins
Delivery of signal from the receptor into the cell is mediated by the action of receptor associated G protein action
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V.W. Rodwell et al., Harper's illustrated biochemistry, 30th edition, pp. 521; © 2015 by The McGraw-Hill Education.
Enzyme-linked receptors
Heterogenous single-pass transmembrane proteins
Receptor functions directly as enzymes or in association with enzymes for signal mediation into the cell
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Alberts et.al., Molecular biology of the cell, 6th edition, pp. 818; © 2015 by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, and Peter Walter.
 General outline of signal transduction via GPCR and
Enzyme linked receptors enzyme linked receptor

Different types of enzyme linked receptors based

on the enzyme used for signal transduction
◦ Receptor guanylyl cyclase e.g., receptor for ANP

◦ Receptor tyrosine kinase e.g., receptors for insulin,

growth factors

◦ Tyrosine kinase associated receptor e.g., receptors

for erythropoietin, growth hormone, cytokines

◦ Receptor serine-threonine kinase

◦ Receptor tyrosine phosphatase

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C.W. Pratt, K. Cornely, Essential Biochemistry, 3rd edition, pp. 270; © 2014, 2011, 2004 John Wiley and Sons, Inc.
Regulation of receptors Receptors number increase or decrease in response to various stimuli

Receptor up-regulation

Number of active receptors decreases

in response to hormone deficiency

Inducing receptor protein gene

expression
Enhancing translocation of receptors to
the membrane

Receptor down-regulation
Number of active receptors increases
in response to hormone excess
Repression of receptor protein gene
expression
Desensitization of receptors
Internalization and degradation of
receptors

37
T. Devlin Ed., Textbook of Biochemistry with clinical correlations, 7th edition, pp. 513; © 2011, 2006, 2002, 1997, 1992, 1986, 1982 John Wiley & Sons, Inc.
Intracellular signal mediators
Molecules involved in transduction of signal G-proteins

received by receptors into the cells

Second messengers such as cAMP, cGMP, Ca2+

Components of intracellular signal

Protein kinases such as PKA, PKC
transduction cascade
Protein phosphatases
Relay signal to the target effector proteins in
order to generate response inside the cell Signal mediator proteins such as calmodulin

Adaptor proteins such as Grb2 protein

DNA binding proteins

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 Signaling molecules that binds to cell Signaling molecules that binds to
surface receptors (hydrophilic signaling intracellular receptors (lipophilic
molecules) signaling molecules)

Signaling molecules that use GPCRs ◦ Steroid hormones (cortisol,

◦ ADH, angiotensin II
aldosterone)

◦ Catecholamines, glucagon ◦ Sex hormones (estrogen,

◦ ACTH, TSH, FSH, LH, TRH, GnRH, testosterone, progestin)

◦ PTH, calcitonin ◦ Thyroid hormone

◦ Vitamin D (calcitriol)
Signaling molecules that use ion-channel
linked receptors ◦ Vitamin A (retinoic acid)

◦ Neurotransmitters

Signaling molecules that use enzyme linked

receptors
◦ Atrial natriuretic peptide (ANP)

◦ Insulin, growth factors

◦ Growth hormones, cytokines, erythropoietin

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D.L. Nelson, M.M. Cox, Lehninger Principles of Biochemistry, 6th edition, pp. 932; ©2013, 2008, 2005, 2000 by W. H. Freeman and Company.
Mechanism of action of lipophilic
signaling molecules

N.V. Bhagavan, Chung-Eun Ha, Essentials of Medical Biochemistry with clinical cases, 2nd edition, pp. 536; © 2011, 2015 Elsevier Inc.
Entry of lipophilic hormones into the target cells

Binding of hormones to the intracellular receptors

Formation of hormone-receptor complex

Serve as intracellular messenger

Acts as gene regulatory protein or specific TF

Binds to hormone response element of specific

genes

Influence the rate of target protein gene

transcription

Alteration in cellular activity

40
Mechanism of action of hydrophilic signaling molecules
(Signaling mechanisms via cell surface receptors)

Cell surface
receptors

Ion channel Enzyme linked

GPCRs
linked receptors receptors

Transient
Tyrosine kinase
opening or Other G proteins Receptor Receptor
Gs/Gi Gq associated
closing of ion- e.g., G12, Gt guanylyl cyclase tyrosine kinase
receptor
channels

Ion permeability
changes and cAMP signaling Phosphoinositide cGMP signaling MAPK signaling JAK-STAT
excitability changes cascade signaling cascade cascade cascade signaling cascade
in target cells

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 Signaling mechanism via ion channel linked receptor
D.L. Nelson, M.M. Cox, Lehninger Principles of Biochemistry, 6th edition, pp. 466; ©2013, 2008, 2005, 2000 by W. H. Freeman and Company.

Signaling mechanism by neurotransmitters

Involved in rapid synaptic signaling between

electrically excitable cells

M. Lieberman, A.D. Marks and A. Peet, Marks' Basic Medical Biochemistry: A clinical approach, 4th edition, pp. 173-
Transient opening or closing of ion channels
associated with receptors upon binding with
neurotransmitters

Conversion of chemical signals from

4; © 2013 Lippincott Williams & Wilkins, a Wolters Kluwer business.

neurotransmitters into electrical potential
changes at synaptic junction

Mediate synaptic signaling by changing the

ion permeability and excitability of the post-
synaptic cells e.g., acetylcholine receptors

42
 Signaling mechanisms via G-protein coupled receptor
Signal received by the receptor is mediated to the effectors through G-proteins

43
D. Sadava et al., Life: The Science of Biology, 10th edition, pp. 130; © 2014 by Sinauer Associates, Inc.
 G protein
GTP binding regulatory proteins that transmits signal
received by receptor to its target enzymes or ion
channels in the cell membrane

Regulate transmembrane signals by acting as molecular

switches, linking cell surface GPCRs to one or more
downstream signaling molecules

Two major sub-families

◦ Small, monomeric Ras-like G proteins (involved in GF and
insulin signaling pathway)

◦ Heterotrimeric G proteins (associated with signal via

GPCRs)

44
J.M. Berg, J.L. Tymoczko, G.J. Gatto, Jr., L. Stryer, Biochemistry, 8th edition, pp. 401; ©2015, 2012, 2007, 2002 by W. H. Freeman and Company; © 1995, 1988, 1981, 1975 by Lubert Stryer.
D.L. Nelson, M.M. Cox, Lehninger Principles of Biochemistry, 6th edition, pp. 440; ©2013, 2008, 2005, 2000 by W. H. Freeman and Company.

L.A. Moron, H.R. Horton, K.G. Scrimgeour and M.D. Perry, Principles of Biochemistry, 5th edition, pp. 286; ©2012, 2006, 2002, 1996
Pearson Education, Inc.
G protein cycle
Active in GTP bound form and regulate the activity of effector proteins (enzymes or ion channels)
α subunit confers effector specificity as well as contains GTP binding site and intrinsic GTPase activity

45
Different classes of
G proteins

Target effector proteins,

second messenger
generated and
subsequent downstream
signaling cascade is
differed by different G
protein types

Major G protein classes

◦ Gs

◦ Gi

◦ Gq

◦ Gt

◦ G12
46
V.W. Rodwell et al., Harper's illustrated biochemistry, 30th edition, pp. 522; © 2015 by The McGraw-Hill Education.
 by type of G protein used for signal mediation
Downstream signaling cascade mediating through GPCRs can be differed

47

D.L. Nelson, M.M. Cox, Lehninger Principles of Biochemistry, 6th edition, pp. 483; ©2013, 2008, 2005, 2000 by W. H. Freeman and Company.
 Effect of cholera toxin on G protein
T. Devlin Ed., Textbook of Biochemistry with clinical correlations, 7th edition, pp. 526; © 2011, 2006, 2002, 1997, 1992, 1986, 1982 John Wiley & Sons, Inc.

Biochemical basis of watery diarrhea in cholera infection

Cholera toxin

ADP • Inhibition of
ribosylation of intrinsic GTPase
Gs-α subunit activity

• Prolonged
Permanent increase in
activation of G cAMP
protein concentration

Opening of Cl− • Excessive water and

channels electrolytes secretion
into GI lumen
• Severe watery diarrhea
48
 Diseases associated with G protein abnormalities

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T.D. Pollard et al., Cell Biology, 3rd edition, pp. 436; © 2017 by Elsevier, Inc.
 Signaling mechanisms via G protein coupled receptors

D.L. Nelson, M.M. Cox, Lehninger Principles of Biochemistry, 6th edition, pp. 483; ©2013, 2008, 2005, 2000 by W. H. Freeman and Company.
50
T. Devlin Ed., Textbook of Biochemistry with clinical correlations, 7th edition, pp. 522; © 2011, 2006, 2002, 1997, 1992, 1986, 1982 John Wiley & Sons, Inc.
Signaling mechanisms via GPCRs

T. Devlin Ed., Textbook of Biochemistry with clinical correlations, 7th edition, pp. 524; © 2011 John Wiley & Sons, Inc.
Downstream signaling cascade
of GPCRs is differed according to
type of G protein used for signal
mediation

Major downstream signaling

cascade via GPCRs

• cAMP signaling cascade

• Phosphoinositide signaling
cascade

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 Intracellular signaling molecules generated upon ligand-receptor interaction and function to mediate
Second messengers signaling effects of hormones in the cells
52
R.H. Garrett and C.M. Grisham, Biochemistry, 6th edition, pp. 1179; © 2017, 2013 Cengage Learning.
Second messengers carry information from cell membrane (receptor) to the target effector protein

Most common second messengers in intracellular signaling cascade are cyclic nucleotides (cAMP, cGMP),
phosphoinositide derivatives (DAG, IP3) and ionized calcium
54
H. Lodish et al., Molecular Cell Biology,, 8th edition, pp. 679; © 2016, 2013, 2008, 2004 by W. H. Freeman and Company.
Signaling mechanism via
cAMP cascade

Hormone binding to GPCR

Activation of Gs protein

Stimulation of adenylyl cyclase

 cAMP concentration

Stimulation of PKA by cAMP

Phosphorylation of effector
protein

Effector protein activity changes

Generation of cellular response

55
D. Voet, J.G. Voet, C.W. Pratt, Fundamentals of Biochemistry, 5th edition, pp. 430; © 2016, 2013, 2008, 2006 by Donald Voet, Judith G. Voet, Charlotte W. Pratt.
D.L. Nelson, M.M. Cox, Lehninger Principles of Biochemistry, 6th edition, pp. 439; ©2013, 2008, 2005, 2000 by W. H. Freeman and Company.

Cyclic adenosine monophosphate (cAMP) is a cyclic nucleotide

Formed from ATP by the action of adenylyl cyclase (AC)
Gs activates adenylyl cyclase and increase cAMP concentration whereas Gi inhibits AC and decrease cAMP concentration
cAMP is degraded to 5’AMP by the action of phosphodiesterase (PDE) 56
cAMP binds and activates cAMP-dependent protein kinase or PKA
PKA phosphorylates and regulates the activity of target effector proteins

D.L. Nelson, M.M. Cox, Lehninger Principles of Biochemistry, 6th edition, pp. 440; ©2013, 2008, 2005, 2000 by W. H. Freeman and Company.
57
Alberts et.al., Molecular biology of the cell, 6th edition, pp. 836; © 2015 by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, and Peter Walter.
 Signaling molecules that use cAMP signaling cascade

V.W. Rodwell et al., Harper's illustrated biochemistry, 30th edition, pp. 521; © 2015 by The McGraw-Hill Education.
58
D.L. Nelson, M.M. Cox, Lehninger Principles of Biochemistry, 6th edition, pp. 446; ©2013, 2008, 2005, 2000 by W. H. Freeman and Company.
 Signaling mechanism of epinephrine via β adrenergic receptor

Example of
signaling
mechanism
of hormone
via cAMP
signaling
cascade

59
D.L. Nelson, M.M. Cox, Lehninger Principles of Biochemistry, 6th edition, pp. 439; ©2013, 2008, 2005, 2000 by W. H. Freeman and Company.
 Signaling
mechanism via
phosphoinositide
cascade

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D. Voet, J.G. Voet, C.W. Pratt, Fundamentals of Biochemistry, 5th edition, pp. 433; © 2016, 2013, 2008, 2006 by Donald Voet, Judith G. Voet, Charlotte W. Pratt.
Signaling mechanism via phosphoinositide cascade

N.V. Bhagavan, Chung-Eun Ha, Essentials of Medical Biochemistry with clinical cases, 2nd edition, pp. 541; © 2011, 2015 Elsevier Inc.
Hormone binding to GPCR

Activation of Gq protein

Stimulation of
phospholipase C (PLC)

Cleavage of PIP2 into IP3 and Translocation of PKC

DAG by the action of PLC towards cell membrane

Opening of IP3 gated Ca2+

channels
Activation of PKC by DAG
and phosphatidylserine
Increased intracellular Ca2+
level

Phosphorylation and
Ca2+-
Activation of Generation of cellular
regulation of target
Activation of calmodulin calmodulin dependent response
protein activity
protein kinase (CaM kinase) 61
Second messengers derived from phospho-inositol diphosphate (PIP2)

Inositol triphosphate (IP3)

• Binds and opens IP3 gated Ca2+

channels on ER

• Results in elevated intracellular Ca2+

• Very short half-life (< a few seconds)

Diacylglycerol (DAG)

• Can be cleaved to release

arachidonic acid which either can act
as second messenger or be used as
precursor in eicosanoid synthesis

• Activates protein kinase C (PKC)

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D. Voet, J.G. Voet, C.W. Pratt, Fundamentals of Biochemistry, 5th edition, pp. 434; © 2016, 2013, 2008, 2006 by Donald Voet, Judith G. Voet, Charlotte W. Pratt.
D. Voet, J.G. Voet, C.W. Pratt, Fundamentals of Biochemistry, 5th edition, pp. 436; © 2016, 2013, 2008, 2006 by Donald Voet, Judith G. Voet, Charlotte W. Pratt.

Signaling effects of ionized calcium

• Downstream signaling cascade of

elevated cytosolic calcium level is
mediated by calcium calcium-
dependent regulatory protein
called calmodulin

• Ca2+-calmodulin complex
regulates the activity of many
structural elements and enzymes
in the cell

• Most of the effects of Ca2+ in cells

are mediated by a family of Ca2+-
calmodulin-dependent protein
kinase (CaM kinase)

63
Signaling molecules that mediate signaling pathway through phosphoinositide cascade

64
D.L. Nelson, M.M. Cox, Lehninger Principles of Biochemistry, 6th edition, pp. 447; ©2013, 2008, 2005, 2000 by W. H. Freeman and Company.
 cascade
signaling
signaling

via cAMP
Example of

mechanism
of hormone

65

T. Devlin Ed., Textbook of Biochemistry with clinical correlations, 7th edition, © 2011 John Wiley & Sons, Inc.
Termination of signaling cascades via GPCR

TERMINATION OF cAMP SIGNALING CASCADE TERMINATION OF CALCIUM SIGNALING CASCADE

Downregulation of GPCR Downregulation of GPCR

Increased rate of deactivation of G protein by Increased rate of deactivation of G protein by

enhancing the intrinsic GTPase activity enhancing the intrinsic GTPase activity

Decreasing cAMP concentration by hydrolysis of cAMP Lowering cytosolic Ca2+ level by:
to 5’ AMP by phosphodiesterases ◦ Sequestration of Ca2+ into ER by Ca2+-ATPase

◦ Chelation with Ca2+ binding proteins such as calsequestrin

Dephosphorylation of target proteins by
phosphoprotein phosphatases Dephosphorylation of target proteins by phosphatases

66
 Signaling mechanisms via enzyme-linked receptors

Major classes of enzyme linked

receptors

• Receptor guanylyl cyclase

• Receptor tyrosine kinase

• Tyrosine kinase associated

receptor

• Receptor serin-threonine kinase

• Receptor tyrosine phosphatase

67
C.K. Mathews, K.E. Van Holde, D.R. Appling, S.J. Anthony-Cahill, Biochemistry, 4th edition, pp. 978; © 2013 Pearson Canada Inc.
Signaling mechanism via receptor guanylyl cyclase or cGMP signaling cascade

Hormone binding to Hormone binding to

receptor guanylyl cytosolic guanylyl

D.L. Nelson, M.M. Cox, Lehninger Principles of Biochemistry, 6th edition, pp. 459; ©2013 W. H. Freeman and Company.
cyclase e.g., ANP cyclase e.g., NO

Activation of guanylyl cyclase activity

 cGMP concentration

Stimulation of cGMP dependent protein

kinase (PKG)

Phosphorylation of target protein

Changes in target protein activity

Generation of cellular response

68
Signaling mechanism of nitric oxide (via cytosolic guanylyl cyclase)

69
H. Lodish et al., Molecular Cell Biology,, 8th edition, pp. 715; © 2016, 2013, 2008, 2004 by W. H. Freeman and Company.
 General steps involved in signaling via
Signaling mechanism via receptor tyrosine kinase RTKs

• Binding of signaling molecule to RTK

• Receptor dimerization

• Activation of tyrosine kinase activity

in cytosolic domain of RTK

• Autophosphorylation and cross-

phosphorylation of specific tyrosine
residues on cytosolic domain of RTK

• Recognition and binding of phospho-

tyrosine residues on cytosolic domain
of receptor by proteins having SH2
Signaling molecules that mediates signaling pathway via RTKs domain
• Insulin • Activation of downstream signaling
• Growth factors such as epidermal growth factor (EGF), platelet derived growth pathway (depends on proteins bound
factor (PDGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), to P-Tyr residues)
insulin like growth factor (IGF-1), nerve growth factor (NGF), vascular endothelial • RAS dependent signaling pathway
growth factor (VEGF) and macrophage colony stimulating factor (M-CSF) • RAS independent signaling pathway
70
T. Devlin Ed., Textbook of Biochemistry with clinical correlations, 7th edition, pp. 517; © 2011, 2006, 2002, 1997, 1992, 1986, 1982 John Wiley & Sons, Inc.
Signaling mechanism via receptor tyrosine kinase Ras dependent Ras independent
signaling cascade signaling cascade

Binding of Grb2-SOS to Binding of enzyme

P-Tyr residues on proteins having SH2
cytosolic domain of domain to P-Tyr
RTK residues e.g., PI3K, PLC

Activation of SOS which Activation of

facilitates GDP to GTP phosphoinositol 3-
exchange in Ras protein kinase (PI3K)

Activation of Ras Phosphorylation of PIP2

protein to form PIP3

Activation of mitogen
Activation of Akt or
activated protein
protein kinase B (PKB)
kinase (MAPK) cascade

Activation of mitogen Initiation of

activated protein downstream signaling
kinase (MAPK) cascade cascade by PKB

71
G. Meisenberg and W.H. Simmons, Principles of Medical Biochemistry, 3rd edition, pp. 302; © 2012, 2006, 1998 by Saunders, an imprint of Elsevier, Inc.
 Growth factor signaling pathway

D.L. Nelson, M.M. Cox, Lehninger Principles of Biochemistry, 6th edition, pp. 487; © 2013 W. H. Freeman and Company.
72

D. Voet, J.G. Voet, C.W. Pratt, Fundamentals of Biochemistry, 5th edition, pp. 413; © 2016 Donald Voet, Judith G. Voet, Charlotte W. Pratt.
Growth factor signaling pathway abnormalities can lead to cancer development
Cancer is strongly associated with the
overactivity of signal transduction proteins in

D.L. Nelson, M.M. Cox, Lehninger Principles of Biochemistry, 6th edition, pp. 498; © 2013 W. H. Freeman and Company.
the growth factor signaling pathway

Abnormal signal transduction pathway leads to

uncontrolled cell growth and cell proliferation

Examples of growth factor signaling pathway

abnormalities associated with cancer
development (products of oncogenes)

• Sis oncogene is growth factor

overexpression (PDGF-β chain)

• Erb-2 oncogene – abnormal growth factor

receptor (EGF receptor)

• Ras oncogene – abnormal Ras protein

activity

• Overactive kinase activity in MAPK cascade

• Overactive cytosolic tyrosine kinases

• Abnormalities in transcription factors such

as fos, jun

73
Signaling mechanism via receptor tyrosine kinase (RTK)

Both growth factors and insulin signaling cascade is

mediated via RTKs. But insulin signaling cascade use adaptor
proteins called insulin receptor substrates (IRS) for
mediation of downstream signaling cascade
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G. Meisenberg and W.H. Simmons, Principles of Medical Biochemistry, 3rd edition, pp. 300-1; © 2012, 2006, 1998 by Saunders, an imprint of Elsevier, Inc.
 Insulin binding to receptor

C.K. Mathews, K.E. Van Holde, D.R. Appling, S.J. Anthony-Cahill, Biochemistry, 4th edition, pp. 979; © 2013 Pearson Canada Inc.
Activation of tyrosine kinase activity

Autophosphorylation and cross-

phosphorylation of tyrosine residues on
cytosolic domain of receptor

Binding of IRS to P-Tyr residues on cytosolic

domain of receptor

Phosphorylation of tyrosine residues on IRS

Binding of proteins having SH2 domain to P-

Tyr residues on IRS

Ras dependent signaling Ras independent signaling

cascade e.g., MAPK cascade cascade e.g., PI3K cascade

Insulin signaling pathway 75

 insulin
General

effects of
outline of

insulin on
target cells
signaling and

76

E.A. Newsholme and T.R. Leech, Functional Biochemistry in health and disease, 2nd edition, pp. 260; © 2010 by John Wiley & Sons, Ltd.
 Signaling mechanism via tyrosine kinase-associated receptor
Tyrosine kinase-associated receptors lack a
D.L. Nelson, M.M. Cox, Lehninger Principles of Biochemistry, 6th edition, pp. 457; © 2013, 2008, 2005, 2000 by W. H. Freeman and Company.

catalytic domain

Depend on non-receptor tyrosine kinase

(soluble TK or cytosolic TK) such as Src and
Janus kinases for their signaling mechanism

Downstream targets of JAKs – transcription

factors called signal transducers and
activators of transcription (STATs)

JAKs also phosphorylate other cytoplasmic

proteins through binding to SH2 domains
e.g., activation of PI3K, MAPK cascade

Hormones that mediate their signaling via

tyrosine kinase-associated receptor

• Growth hormone, prolactin,

erythropoietin, cytokines, and antigen-
specific receptors on T and B
lymphocytes
77
 Overview of
signaling
pathways via
cell surface
receptors

79
C.K. Mathews, K.E. Van Holde, D.R. Appling, S.J. Anthony-Cahill, Biochemistry, 4th edition, pp. 961; © 2013 Pearson Canada Inc.
Overview of signaling pathways via cell surface receptors and cross-talk between signaling pathways

D.L. Nelson, M.M. Cox, Lehninger Principles of Biochemistry, 6th edition, pp. 458; ©2013, 2008, 2005, 2000 by W. H. Freeman and Company.

Alberts et.al., Molecular biology of the cell, 6th edition, pp. 862; © 2015 by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin 81
Raff, Keith Roberts, and Peter Walter.
Signal termination mechanisms

Negative feedback control on secretion of signaling molecules

Degradation and clearance of signaling molecules

Down-regulation of receptors

Termination of intracellular signal mediators action

◦ Accelerated inactivation of G protein

◦ Degradation of second messengers e.g., degradation of cAMP, and

cGMP by specific phosphodiesterases (PDEs)

◦ Lowering cytosolic Ca2+ level

◦ Degradation of intracellular signal-mediating proteins

◦ Reversing the action of protein kinases through dephosphorylation

of target protein by the action of protein phosphatases
82
M. Lieberman, A.D. Marks and A. Peet, Marks' Basic Medical Biochemistry: A clinical approach, 4th edition, pp. 187; © 2013 Lippincott Williams & Wilkins, a Wolters Kluwer business.
Signal termination mechanisms
83
Alberts et.al., Molecular biology of the cell, 6th edition, pp. 831; © 2015 by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, and Peter Walter.
Mechanisms of signaling pathways
affecting the rate of specific gene

T.D. Pollard et al., Cell Biology, 3rd edition, pp. 472; © 2017 by Elsevier, Inc.
expression in target cells

Hormonal regulation of gene expression

84
Signaling pathways of
special senses
SIGNALING MECHANISM IN VISION

S I G N A L I N G M E C H A N I S M I N O L FA C T I O N

S I G N A L I N G M E C H A N I S M I N G U S TAT I O N

85
 Signaling mechanism in vision

86
D.L. Nelson, M.M. Cox, Lehninger Principles of Biochemistry, 6th edition, pp. 477, 479; ©2013, 2008, 2005, 2000 by W. H. Freeman and Company.
 Signaling mechanism
in olfaction

87
D.L. Nelson, M.M. Cox, Lehninger Principles of Biochemistry, 6th edition, pp. 482; ©2013, 2008, 2005, 2000 by W. H. Freeman and Company.
Signaling mechanism in gustation (Gustatory signaling)

88
D.L. Nelson, M.M. Cox, Lehninger Principles of Biochemistry, 6th edition, pp. 482; ©2013, 2008, 2005, 2000 by W. H. Freeman and Company.
D.L. Nelson, M.M. Cox, Lehninger Principles of Biochemistry, 6th edition, pp. 436; © 2013, 2008, 2005, 2000 by W. H. Freeman and Company.

pathways and signal

Overview of signaling

transduction mechanisms

89
References
Alberts, B., Heald, R., Johnson, A., Morgan, D., Raff, M., Roberts, K., Walter, P., Wilson, J., and Hunt, T. (2022) Molecular Biology of the Cell, 7th edition. New York, NY. © Bruce
Alberts, et al.

Iwasa, J., and Marshall, W. (2016) Karp’s Cell and Molecular Biology: Concepts and Experiments, 8th edition. Hoboken, NJ. © John Wiley & Sons, Inc.

Lieberman, M., and Peet, A. (2022) Mark’s Basic Medical Biochemistry A Clinical Approach, 6th edition. Baltimore, MD. © Wolters Kluwer.

Lodish, H., Berk, A., Kaiser, C.A., Krieger, M., Bretscher, A., Ploegh, H., Martin, K.C., Yaffe, M.B., and Amon, A. (2021) Molecular Cell Biology, 9th edition. New York, NY. © W. H.
Freeman and Company.

Miesfeld, R.L., and McEvoy, M.M. (2017) Biochemistry, 1st edition. New York, NY. © W. W. Norton & Company, Inc.

Nelson, D.L., Cox, M.M., and Hoskins, A.A. (2021) Lehninger Principles of Biochemistry, 8th edition. New York, NY. © W. H. Freeman and Company.

Newsholme, E.A., and Leech, T.R. (2010) Functional Biochemistry in health and disease, 2nd edition, © John Wiley & Sons, Ltd.

Pratt, C.W., and Cornely, K. (2018) Essential Biochemistry, 4th edition. Hoboken, NJ. © John Wiley and Sons, Inc.

Salt, I.P. (2019) Chapter 25: Membrane receptors and signal transduction, In: Baynes, J.W., and Dominiczak, M.H. (eds.) Medical Biochemistry, 5th edition. Philadelphia, PA. ©
Elsevier Limited.

Weil, P.A. (2022) Chapter 42: Hormone action & signal transduction, In: Kennelly, P.J., et al. Harper’s Illustrated Biochemistry, 32nd edition. New York, NY. © McGraw Hill, LLC.

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