regulation of intracellular calcium signaling, localized signals … · 2015-12-25 · in...

Regulation of Intracellular Calcium Signaling,

Localized Signals and Oscillations

Prof. Barbara E. Ehrlich

The screen versions of these slides have full details of copyright and acknowledgements 1

1

Regulation of Intracellular

Calcium Signaling, Localized Signalsand Oscillations


Yale University

Pharmacology and Cellular and Molecular PhysiologyLaboratory of Molecular Hermeneutics

Summer 2006

2

• Introduction to calcium waves and oscillations

• Introduction to calcium signaling

• Signal initiation in dendrites (the effect of chromogranin)

• Signal initiation in the cytoplasm(stimulation by testosterone)

• Regulation of oscillations (action of protein tyrosine phosphatase)

Outline of Lecture

3

What Are Calcium Waves and Oscillations?

SHSY5Y cells

Fluo-4 loaded

Waves = changes in space

Oscillations = changes in time





4

0

1

2

3

0 30 60 90 120 150 18 0

tim e (s )

F/F0

50 µMCarbachol

Black = somaGray = neurite

Isolated hippocampal cell in culture

Starts in the soma

Neuronally-differentiated PC12 cell

Starts in the neurite

The initiation site for a calcium wave is cell specific

Calcium Waves

8 mV2 s

10% ∆∆∆∆ F/F0

Vn

5

Calcium Oscillations

Epithelial cells

INS-1 cells

Time (s)

Neuronal cells

SHSY5Y cells

Time (s)

6

The Frequency of Calcium TransientsEncodes Important Information

Dolmetsch, Xu and Lewis, 1998

Fidelity

Specificity

Oscillations

Spontaneous

(e.g., cardiomyocytes)Induced (e.g., epithelia)

The frequency can be modulated





7

Changes in Intracellular Calcium SignalingOccur in Many Diseases

Huntington’s disease

Alzheimer’s disease

Parkinson’s disease

Schizophrenia

Heart failure

Bile duct obstruction

Polycystic kidney disease

8

Outline of Lecture






9

What Is Needed for Intracellular Calcium Signaling?





10

InsP3R Type I Are Concentrated in Cerebellar Purkinje Cells

Rat cerebellar slice [12 days PN]; Polyclonal antibody for type I InsP3 receptor, Llano and Ehrlich

11

InsP3 Receptor:Three Isoforms and Three Domains

Patel et al., Cell Calcium, 25: 247 (1999)

12

The InsP3R Is a Large Protein that Makes an Ion Channel

250

100

50

kDa

Popen

0%

2%Closed

Closed

Open

Open





13

InsP3 Production Is Required

Expression of a lipid phosphatase (SJ-1) inhibits calcium release

Inhibit response

Control

Inhibit response

SJ-1 expression

1234

14

The InsP3 Dependence Is Differentfor Each of the InsP3R Isoforms

In the presence of multiple InsP3R isoforms,

the type I, then type III will respond to InsP3 production

InsP3 (mM)

15

Ca2+ Dependence of the InsP3R Isoforms

In the presence of multiple InsP3R isoforms, cells with the type I

will oscillate, cells with the type II and III may not

Ca2+ (mM)





16

Outline of Lecture






17

Chromogranin A and B Bind to the InsP3R

Chromogranin A (CGA) and B (CGB) are high-capacity, low-affinity Ca2+

storage proteins

They bind 50–100 mol Ca2+/mol Chromogranin with dissociation constants of 2-4 mM

They are found in secretory granules; CGB also in ER of many cell types

N-terminal conserved region in CGA and CGB interact with L3-2 of the InsP3R

Yoo, S.H., TINS (2000)

17 38 407 431

16 37 602 628

18

Can Calcium Buffers Associate with the InsP3R and Regulate Channel

Activity from the Lumenal Side?





19

Effect of Chromogranin Bon InsP3R Activity

5%

80%

40%

pH 5.5

pH 5.5+CGB

pH 7.5+CGB

PoLumenal pH

closedopen

closedopen

closedopen

20

CGB Fragment AltersInsP3R Channel Function

16 37 602 626

21

CGB Fragment AltersCalcium Release in PC12 Cells

CGB-fragment

Calnexin

Overlay





22

Chromogranin B

The Localization of CGBIs Not Uniform in Cells

neuronally-differentiated PC12 cell

It could be a factor in determining the signal initiation site

Isolated hippocampal cell in culture

23

Parvalbumin SERCA 2

Many Other Proteins Are Uniformly Distributed

Two examples of proteins which are importantfor down regulation of calcium levels

24

The Functional Coupling of CGBto the InsP3R Can Play a Role

in Determining the Signal Initiation Site

CGB-fragment

calnexin

overlay

Control CGB fragment

Tim

e (s)

dt (soma-neurite) for 500µM carbachol

P < 0.001





25

InsP3 R 1

CA3 CA1

CGB

CA1CA3

The Localization of CGB Is Not Uniformin Cells of the Rat Hippocampus

26

Chromogranin B levels are lower in brain tissue

from patients with schizophrenia

Control Schizophrenia

Cell body

Dendrites

Nowakowski et al., Schizophrenia Research 58 (2002) 43-53

Disease-Related Changes in Calcium Signaling

27

Conclusions: InsP3R and Chromogranin

• Chromogranin regulates the InsP3R from the lumen of the ER

• Chromogranin is concentrated in specific cell regions and in specific cell types

• High concentrations of chromogranin correlate

with signal initiation sites

CGB





28

Outline of Lecture






29

Testosterone Induced Calcium Oscillations

30

The Importance of Buffers





31

Calcium Buffers Can Be Expressed in Specific Regions of a Cell

Propidium iodide MergeGFP

PV-N

LS-GFP

PV-N

ES-GFP

32

Testosterone Induced Calcium OscillationsCytoplasmic and nuclear signals can be separated

33

Testosterone Induced Neurite OutgrowthCytoplasmic and nuclear signals can be separated

A B

PV-NES-DSR

PV-NLS-DSR

PV-NLS-CDEF-DSR





34

High Testosterone Decreases Cell Viability

35

High Testosterone ChangesThe calcium signal

36

Conclusions: InsP3R and Testosterone

• Testosterone induces calcium

oscillations independent of the iAR

• Testosterone induced calcium oscillations begin in the cytoplasm

• Low concentrations of testosterone enhance neurite growth

• High concentrations of testosterone induce cell death (apoptosis)

Plasma membrane

Cytoplasm





37

Outline of Lecture






38

SHP-2: SH2 Domain-Containing Protein Tyrosine Phosphatase

• Ubiquitously expressed protein tyrosine phosphatase containing two src homology 2 (SH2) domains

• SHP-2 is required for positive regulation of receptor tyrosine kinase (RTK) signaling

• Binds activated growth factor receptors directly (e.g., EGF, PDGF) and/or RTK scaffold proteins (e.g., FRS-2)

• Mutations in SHP-2 cause human developmental disorder called Noonan syndrome (1:1,000-1:2,500 live births)

39

SHP-2 Positively Regulates RTK Pathways

SHP-2Ex3-/-

SHP-2 functional Knock-out Has defectiveRTK signaling





40

SHP-2 Is Required for Fibroblast Growth Factor (FGF)-Induced Calcium Oscillations

SHP-2Ex3-/-SHP-2+/+

41

Noonan’s SyndromeAutosomal dominant disorder characterized by:

• dysmorphic facial features

• heart disease (pulmonic stenosis

and hypertrophic cardiomyopath y)

• webbed neck

• mental retardation

1:1,000-1:2,500 live births

Mapped to a 5 cM region (N-SH2)

on chromosome 12q24.1

Mutations in PTPN11: a gene encoding

the protein tyrosine phosphatase SHP-2 – cause Noonan syndrome

Noonan syndrome mutations result in enhanced tyrosine phosphatase activity

Hum Genet (2002) 111: 421–427

42

Noonan Syndrome Mutation of SHP-2 Enhances Calcium Oscillatory Frequency





43

SHP-2 Modulates Spontaneous Calcium Oscillations in Cardiac Myocytes

44

Frequency of Calcium Oscillationsand NFAT Activity

Vector (green circle)SHP-2R465M (blue circle)SHP-2WT (yellow circle)SHP-2E76A (red circle)

NFAT activity

(%)

45

Conclusions InsP3R and SHP-2

• SHP-2 is required for FGF-

and PDGF- induced calcium signaling

• SHP-2 (Noonan mutation) enhances the frequency of calcium oscillations

• SHP-2 (Noonan mutation) impairs NFAT signaling





46

Conclusions• The initiation site for calcium signals is cell specific

• The initiation site for calcium signals can be modulated

• Calcium oscillations encode information which is specific

• Calcium oscillations can be induced and modulated

• Calcium oscillations are used to regulate many cell processes

47

GeorgiaAnyatonwu

Juliana Rengifo

Craig Gibson

Eva Winkler Felix Heidrich

DavidJohnston

Wolfgang Boehmerle

Chi-un Choe

Simon Jacob

Nils Nicolay

Per Uhlen

FriedrichJohenning

ManuelEstrada

AnuragVarshney

Brenda DeGray

ChristinaSchlecker

Andjelka Celic

Collaborators

Fred SigworthQiu-Xing Jiang

Steve SomloYiqiang Cai

Anton BennettWayne Grant

MarkYeckel

Mike NathansonFatima Leite

DanielHertle

David Chester

Waves+ CGB

TestosteroneSHP-2

48

regulation of intracellular calcium signaling, localized signals … · 2015-12-25 · in...

Documents