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2005-01-24 19:28:01 | Hit : 26483 | Vote : 7696

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Cell Signaling

Cell Signaling

A. Introduction

1. All cells received and respond to signals from their surroundings
1) bacteria: nutrients
2) unicellular eukaryotes: cell to cell communication (ex. mating between yeast)
3) multicellular organism: complex (by a varierty of signaling molecules)

2. receptor recognition initiates a series of intracellular reactions that regulate virtually  all aspects of cell behavior (metabolism, movement, proliferation, differentiation)
3. Understanding the molecular mechanisms responsible for these pathways of cell signaling has thus become a major area of active research.
4. Interest in this area is further heightened by the fact that most cancers arise as a result of a breakdown in the signaling pathways that control normal cell growth and differentiation.

B. Sinaling molecules and their receptors

Mode of Cell-Cell Signaling

1. endocrine signaling: endocrine cell→hormone→circulation→target cell
2. paracrine signaling: neighboring target cells (neurotransmitters, during embrionic development)
3. autocrine signaling: - cytokine (Interukin II),
                           - abnormal autocrine siganling→cancer

Steroid hormones and the steroid receptor superfamily

1. steroic hormone (small hydrophobic signling molecules)
1) gonad: testostterone, estrogen, progesterone (sex hormone)
2) adrenal gland: glucocorticoids, mineralocorticoids
3) thyroid gland: thyroid hormone
4) vitamin D3, retinoic acid

2. receptor: in the cytosol or the necleus
1) steroid receptor: transcription factors: ligand binding domain
                                DNA binding domain
                                transcription activation domain

Nitric Oxide
1. major paracrine signaling molecule
2. diffusible
3. synthesis: Nitric oxide synthase (arginine→nitrix oxide + citrulline)
4. local effects (unstable)
5. Ach→NO→cGMP→blood vessels dilation
6. nitroglycerin: treatment of heart disease.

Neurotransmitters
1. 종류: acetylcholine, glycin, glutamate, dopamine, norepinephrine, epinephrine, serontonin, histamine, GABA
2. hydrophilic molecules
3. receptor: 1) ligand-gated ion channels (acetylcholine)
            2) G-protein coupled receptor

Peptide Hormones and Growth Factors
1. the widest variety of signaling molecules in animals
2. peptide hormones: insulin, glucagon, pituitary gland hormone
3. neuropeptide: enkephalins, endorphins (neurotransmitters, neurohormone)

4. growth factor: nerve growth factor(NGF), epidermal growth factor(EGF), platelet-drived growth factor(PDGF)
5. cytokines: development and differentiation of blood cells
             activity of lymphocytes during immune response
6. receptor: on the plasma membrane

Eicosanoids
1. lipid
2. binding to cell surface receptor
3. prostaglandins, prostacyclin, thromboxanes, leukotrienes.
4. rapidly  broken down
5. act locally in autocrine or paracrine signaling pathway.
6. response: blood platelet aggregation, inflammation, smooth-muscle contraction
7.  arachidonic acid로부터 생성
8. prostaglandine synthase: target of aspirin
9. aspirin: thromboxane 합성억제→platelet aggregation and blood  clotting 감소→심장마비억제

C. Functions of Cell Surface Receptors

G Protein-Coupled Receptor (Seven Transmembrane Receptor)
1. G protein-coupled receptor를 경유하여 signal를 보내는 hormone
  1) neurotransmitter: acetylcholine, epinephrine
  2) neutopeptide
  3) peptide hormone: oxytocin, vasopressine
2. G protein (guanine nucleotide binding protein)
  1) tetramer (αβγ)
  2) 다양한 isoform 존재: 16α, 5β, 11γ
  3) αsubunit: GTP 혹은 GDP와 결합
               GTPase activity 갖고 있음

3. 다양한 G protein의 다양한 기능.
  1) epinephrine, glucagone, serotonine
      ◆ in liver
     Gsα→ adenylyl cyclase→cAMP 증가→glycogen breakdown  → increase in glucose in blood
        ◆ in muscle
     Gsα→ adenylyl cyclase→cAMP 증가→glycogen breakdown  → increase in glycolysis → ATP 합성
        ◆ in heart
     Gsα→ adenylyl cyclase→cAMP 증가→Ca2+ 증가 → hear contraction 증가
  2) M1, 3, 5 receptor (fig. 13.24)
     Gqα→ phospholipase C-β → IP3 → Ca2+→ 근육수축
                          ↓
                          diacylglycerol → PKC
  3) M2 receptor (심장 부교감신경→Ach→M2 receptor)
     Giα → K+ channel open → hyperpolarization → slowing heart contraction
  4) M4 receptor
     Giα → adenylate cyclase 억제

Receptor Protein-Tyrosine Kinases (RTK)

1. RTK: 대부분의 polypeptide growth factor receptor
2. tyrosine kinase activity 갖고있음
3. animal cell growth와 differentiation에 관여함
4. protein-tyrosine phosphorylation as a key signaling mechanism in  the response of cells to growh factor stimulation
5. 모든 RTK는 공통적인 구조를 갖고 있다 (fig. 13.13).
  1) N-terminal extracellular ligand-binding domain
  2) a single transmembrane αhelix
  3) a cytosolic C-terminal domain with protein-tyrosine kinase activity
6. RTK: 대부분 single polypeptide (예외: insulin receptor:4subunits)
7. RTK의 작용기전 (fig. 13.15)
  ligand binding to RTK→dimerization→autophosphorylation(cross phosphorylation)→ phosphorylation of tyrosine residue→SH2   domain을 갖고 있는 signaling moleculed의 binding site 제공
8. SH2 domain
  1) 약 100개의 amino acid로 구성
  2) bind to specific short peptide sequences containing phsophotyrosine residues

Cytokine Receptor and Nonreceptor Protein-Tyrosine Kinases

1. cytokine receptor:
1) enzyme activity는 없지만 noncovalently associated intracellular protein-tyrosine kinase를 활성화한다
2) 구조: RTK와 유사 (예외: no tyrosine kinase activity)
2. cytokine receptor의 작용기전 (fig. 13.17)
ligand binding to receptor→dimerization→autophosphorylation of the  nonreceptor tyrosine kinase → phosphorylation of receptor →   phosphorylated tyrosine residue→SH2 domain을 갖고 있는 signaling  moleculed의 binding site 제공
3. nonreceptor tyrosine kinase: two major families (Src family/JAK family)
1) Src family
  - src: oncogenic protein of Rous sarcoma virus
  - src family: 8 closely related protein
  - B and T lymphocytes의 antigen receptor로부터의 signaling에 중요한 역할담당
2) Janus kinase (JAK, family)
  - 대부분의 다른 cytokine receptor의 signaling에 관여하여 중요한 역할

Receptors Linked to Other Enzymatic Activities

1. protein-tyrosine phosphatases: CD45 on the surface of T and B cells
2. transforming growth factor β:
- RTK와 유사한 구조
- heterodimer
- serine/threonine kinase
- inhibit proliferation of target cells
3. receptor guanylyl cyclase
- RTK와 유사한 구조
- guanylyl cyclase activity
- cGMP 생성

D. Pathways of Intracellular Signal Transduction

intracellular signal transduction의 기능
◆ propagation and amplication of the signal initiated by ligand binding
◆ connect the cell surface to the nucleus, leading to changes in gene expression in response to extracellular stimuli

The cAMP Pathway: Second Messengers and Protein Phosphorylation

1. epinephrine에의한 glycogen breakdown (fig. 13.20)
2. activation of transcription by cAMP (fig. 13.21)
  ◆ PKA↑→ phosphorylation of CREB(cAMP response element binding protein)→ binding to CRE → activation of transcription
3. cAMP can directly regulate inon channels
  ◆ odorant receptors in sensory neurons in the nose
    cAMP→Na+ channels open→depolarization→inhibition of a nerve impules

Cyclic GMP

1. guanylyl cyclase에의해 생성
2. nitric oxide and peptide ligand activate guanylyl cyclase
3. cGMP:
1) blood vessel dilation (정확한 mechanism은 아직 밝혀지지 않았음)
2) role in the vertebrate eye
  light→rhodopsin(G protein-coupled receptor)→isomerization of 11-cis-retinal to all-trans-retinal→binding to transducin(G protein)→cGMP phosphodiesterase→decrease in cGMP→translation to nerve impulse by a direct effect of cGMP on ion channels.

Phospholipids and Ca2+

1. PLC-β와 PLC-γ에 의한 second messengers 생성 (fig. 13.24, 13.25)
1) diacylglycerol (DG)
   ◆ tumor promotion
     diacylglycerol (DG)→PKC→MAP kinase pathway→transcription  factor phosphorylation→changes in gene expression→cell proliferation (tumor promoter PMA)
   ◆ immune response (fig. 13.27)
2) inositol 1,4,5-trisphosphate (IP3)
3) IP3에의한 Ca2+ mobilization (fig. 13.28)
    - many of the effects of Ca2+ are mediated by the calmodulin (fig.13.29)
    - myosine light-chain kinase
    - CaM kinsase II
    - direct effect of Ca2+ in nerve and muscle (fig. 13.30)
2. phosphatidylinositide 3-kinase (PI3K)
1) SH2 domanin 함유
2) RTK에의해 활성화
3) PI3K의 product: phosphatidylinositol 3,4,5-triphosphate

Ras, Raf, and MAP kinase Pathway

1. Growth factor signal transduction pathway중에서 가장 잘 알려진 pathway
2. RTK→Grb2/SOS→Ras·GTP→Raf→MEK→ERK→Elk-1→transcription
3. Ras
- small G protein
- oncogenic proteins of tumor virus that cause sarcomas in rats
- mutation of human ras gene이 human cancer의 발생에 관여
- dominant negative or positive Ras, antibody microinjection에 의해 역할 규명
- guanine nucleotide exchange factors에의해 활성화
- mutated Ras protein은 GTPase activity가 억제되어 있음
4. Grb2 and SOS
- Grb2: SH2 domain을 갖고 있는 adaptor molecule
- SOS: guanine nucleotide excnange factors
- Grb2와 SOS는 cytosol에서 binding한 상태로 존재
- RTK의 활성화시 RTK에 binding 하여 cytosol로부터 membrane으로 이동한다.
- 그곳에서 membrane에 위치하고 있는 Ras·GDP를 Ras·GTP로    활성화한다
5. Raf
- Ras·GTP는 cytosol에 존재하고 있는 Raf와 binding하여 Raf를  membrane으로 이동시켜 활성화 한다.
- Raf는 MEK를 다시 활성화 한다
6. MEK, ERK
- MEK에의해 활성화된 ERK는 nucleus로 이동하여 transcription factor인 Elk-1를 phosphorylation하여 phosphorylated Elk-1은 serum response element (SRE)에 binding하여 immediate early genes (50-100 genes)를 induction한다.

Cancer, Signal Transduction, and the ras Oncogenes

The Disease
The common feature of all cancers
- the unrestrained proliferation of cancer cells,
- which eventually spread throughout the body,
- invading normal tissues and organs and
- leading to death of the patient.
The currently available chemotherpeutic agents
- are not specific for cancer cell
- act by either damaging DNA or interfering with DNA synthesis.
- Therefore, they also koll rapidly dividing normal cells, such as the epithelial cells that line the digestive tract and the blood-forming    cells of the bone marrow.
- Consequently, although major progress has been made in cancer treatment, nearly half of all patients diagnosed with cancer ultimately    die of their disease.

Molecular and Cellular Basis
ras oncogene:
1) first identified cellular oncogene in human bladder, lung, and colon cancers.
2) one of the most common genetic abnormalities in human tumors
3) found in about 15% of all human cnacers
  25% of lung cancers
  50% of colon cnacers
  90% of pancreatic cancers
4) linked the development of human cancer to abnormalities in the signaling pathways that regulate cell proliferation
5) decrease GTP hydrolysis by the Ras proteins
6) remain locked in the active GTP-bound form
7) thus continuously stimulate the MAP kinase pathway and drive cell proliferation, even in the absence of the growth factors

Prevention and Treatment
1. The discovery of mutated oncogenes in human cancers raises the possibility of developing drugs specifically targeted against the oncogene      proteins.
2. In principle, such drugs mignt act selectively against cancer cells with less toxicity toward normal cells than that of conventional chemotherapeutic agents.
3. Ras-targented drugs: farnesyl transferase inhibitor
  ◆ display substantial selectivity in their action against tumor cells expressing oncogenic Ras protein

The JAK/STAT Pathway

1. JAK/STAT pathway provides a much more immediate connection between protein-tyrosine kinases and transcription factors, while the MAP kinase pathway provides an indirect connection between the cell surface and the nucleus (fig. 13.37)
   STAT(Signal Transducers and Activators of Transcription)
2. STAT proteins
1) a family of transcription factors
2) SH2 domain
3) localized to the cytoplasm in unstimmulated cells
4) binding to the receptors
5) phosphorylation at tyrosine residue by JAK
6) dimerization of STAT proteins
7) translocation to the nucleus
8) stimulation of transcription of their target genes
9) also activated by RTK or norn receptor kinase associated with RTK
10) thus serve as direct links between both cytokine and growth factor receptors on the cell surface and regulation of gene expression in     the nucleus

E. Signal Transduction and The Cytoskeleton

1. The functions of most cells are also directly affected by cell adhesion and the organization of the cytoskeleton.
2. The receptors resposible for cell adhesion thus act to initiate intracellular signaling pathways that regulate other aspects of cell behavior, including gene expression
3. Growth factors frequently act to induce cytoskeletal alterations resulting in cell movement or changes in cell shape.

Integrins and Signal Transduction

1. Integrin
1) structural role
  - the major receptors responsible for the attachment of cells to the extracellular matrix
  - focal adhesions and hemidesmosomes에 관여 (fig. 12.61)
2) role as receptor
  - activate intracellular signaling pathways, thereby controlling gene expression
  - activate other aspects of cell behavior in response to adhesive interactions (fig. 13.38)
3) 구조적 특징
  - short cytoplasmic tails
  - no enzymatic activity
  - associated with nonreceptor protein-tyrosine kinases,
    focal adhesion kinase (FAK)
4) signal transduction
  binding of integrin to extracellular matrix (fibronectin)→tyrosine   phosphorylation of FAK→binding of src to FAK→     phosphorylation additional site on FAK→binding of downstream   signaling molecules containing SH2 domain (PI3K or Grb2-Sos   complex)→changes in gene expression and cell behavior

Regulation of the Actin Cytoskeleton

1. Growth factors frequently cause changes in cell movement and cell shape
2. Growth factor-induced alterations in cell motility (as well as cell proliferation) play critical roles in processes (wound healing, embrionic development).
3. these aspects of cell behavior are governed by the actin cytoskeleton
4. Many types of cell movement are based on the dynamic assembly and disassembly of actin filaments underlying the plasma membrane
5 Rho subfamily of small G protein
- Cdc42→filopodia
- Rac→lamellipodia, membrane ruffles
- Roh→focal adhesions, stess fivers
6. Function of Rho subfamily
1) as regulator of gene expression by activation of MAP kinase signaling pathway
2) as regulator of cytoskeletal remodeling
7. Cdc42→Rac→Rho
- These proteins coordinately control the cytoskeletal alterations responsible to cell movement
- The crawling movements of cell across a surface can be viewed as a series of three types of events (fig. 11.30)