Cellular Biology

1. Cellular Biology School of Life Sciences Shaanxi Normal University 1

2. CHAPTER 7 CELL COMMUNICATION

3. Transportation: (See your text book) 70% energy of cell will be used for transportation. There are two types of transproteins: Carrier protein (carrier, permease, transporter) Channel protein Three types of transportations: Free diffusion (non-polarized molecules) Passive transportation Facilitated diffusion (polarized molecules) Co-transportation Na+-K+ ATPase (Pump) Automatic transportation Proton pump Ca+ pump ABC transporter Endocytosis and exocytosis

4. Roles of cell communication

5. I. Basic concepts Some concepts that are easy to be confused: Now, there are many terms about cell communication used in cell biology, especially to the cell biology of tumor. But, some of them are easy to be confused. I define them as the follows: Cell signaling: Cells release some signal out to some cells else. Cell communication: The signal from a cell is transmitted to another cell by some transmitter, and causes a specific reaction. Cell recognition: A cell interacts with another cell by the signal molecules located on cell surface, and causes specific response of another cell. Signal transduction: The signals from out side of cell (Light, electricity, and molecules) are received by the receptors located on cell surface, cause the change of intracellular signal level, and start a serial responses of cell.

6. Signal molecules: Signals can be chemical signals or physical signals. Chemical signal is much more important than physical signal in cell biology. Chemical signals include oligopeptides, proteins, gas molecules (NO、CO), amino acids, nucleotides, lipids, cholesterol, and others. The characters of chemical signals are ① specificity; ② efficiency. One or more molecules can cause a strong response because the signal transduction system can enlarge the signal stimulation; ③ they can be inactivated after the signal transmission. This is a protection mechanism that organs obtained during the evolutionary history. By the routes of generation and role, the chemical signal molecules can be sorted as 4 types: hormones, neuron transmitters, local mediated factors, and gas molecules. By the solubility, the signal molecules can be sorted as two types: lipid soluble and water soluble molecules. Lipid soluble signal, such as some hormone, can be transported into cell directly passing through the bilayers membrane. Water soluble signal molecule, such as neuron transmitters, can not pass through the bilayers membrane. They have to bind to their receptors and exchange the signal type, transfer the information to the intracellular signal (cAMP) or activate the kinase for receptor to result in cell responses. So, we call these signal molecules as primary messenger, and intracellular signal molecules (cAMP, cGMP, IP3 and DG) as secondary messenger.

7. Ca2+ can be named as third messenger because its signal transmission is depended on secondary messenger. Secondary messenger can enlarge the signal function. Receptors: Receptor can selectively bind to its ligand (Signal molecules). Receptor is glycoprotein usually composed of two function domains at least (ligand binding part and activating part). The features of the interaction between ligand and receptor are: ① specificity; ② saturated limit; ③ high affinity. By the receptor’s location, we can sort receptors as intracellular receptor and cell surface receptor. Intracellular receptor receives lipid soluble signal, and cell surface receptor receives water soluble signal. The response of cell to a signal depends on both receptor and cell type. Same signal can cause different responses on different cells. For examples, Ach can cause the contraction of skeleton muscle, inhibit the contraction rate of heart muscle, and cause the secretion of saliva. Different signal can cause same responses also. For examples, both adrenalin and pancreatic glucagon can enhance the level of blood sugar. If some signal stimulates cell consistently, cell can make its receptor obtuse by the ways as following: ① modify and inactivate receptor. ② move the receptor into inside of cell (receptor sequestration). ③ By endocytosis, digest receptor with lysosome (receptor down-regulation).

8. Cell surface receptor and intracellular receptor

9. Protein Kinase Protein kinase is a type of phosphate transferase that can transfer the Pi from ATP to specific amino acid residue to phosphate and activate protein. The functions of protein kinase during the signal transduction include: 1. regulate protein activity by phosphorylation. Some proteins will be activated by phosphorylation, and some will be inactivated by this modification. 2. enlarge the signal responses by the phosphorylation. Types of protein kinase

10. Communication types among cells There are three main types for the communication. Communication by gap junction: By gap junction, cells can communicate each other directly based on connexons that are tubes with a hydrophilic tunnel at 1.5nm diameter inside. Connexons allow micromolecules, such as, Ca2+, cAMP passed through, that enhances the same type and bordered cells to response to the signals from other cells, such as, electric excitation.

11. Gap junction

12. The communication by plasma membrane bound molecules: We can call this communication as cell recognition that means the interaction between receptors and their ligands. We can sort the recognitions as the following types: ① The recognition between same type cells from same species of animals. For examples, cell can recognize the bordered cells during the development of embryo. The reactions of blood transmission and skin transplantation are the recognition between same type cells from different resource. ② The recognition between different cells from same species. For examples, sperm and ovum, T cell and B cell. ③ The recognition between different cells from different species. For example, pathogens and host cells. ④ The recognition between same type cells from different species. This recognition can be managed under experimental condition.

13. Chemical communication: Chemical communication is the indirect communication of cells. This communication means that cells secret (signaling) the signal molecules, such as, hormone, and excite the target cells to regulate the functions of the target cells. 1. Endocrine: Hormone can regulate the function of target cells distributed anywhere inside of body efficiently, systemically, specifically, and consistently. 2.  Paracrine: The signal molecules secreted by cells can spread to the neighbored cells to regulate them. For example, cytokines and gas molecules (NO). 3.  Synapse signaling. 4.  Autocrine: The signaling cell and target cell are same type, or same one (signaling and targeting itself). Autocrine exists in cancer cells usually. For example, Colic cancer cells can secrete gastrin to mediate the expression of oncogenes (c-myc, c-fos, ras, p21, and others) for the enhancement of tumor growth.

14. Chemical communication

15. The types of chemical communication

16. II. The signal transductions mediated by plasma membrane bound receptors Hydrophilic signal molecules (neuron transmitters, hormones, growth factors, and others) can not enter cell directly, they must combine to the specific receptors bound on membrane surface to cause cell responses. The membrane surface bound receptors can be sorted as three types as the follows: ① Ion-channel-linked receptor; ② G-protein-linked receptor; ③ Enzyme-linked receptor. Ion-channel-linked receptor distributes on excitable cells. G-protein-linked receptor and enzyme-linked receptor are located on most of cell types that can work as kinase cascade to phosphate proteins with a serial phosphorylation reactions to transfer and enlarge the signal step by step.

17. Three types of membrane surface bound receptors

18. Ion-channel-linked receptor: Ion-channel-linked receptor is a ligand-gated channel receptor distributed on excitable cells, such as, nerve and muscle cells using neuron transmitters as signal molecules. Neuron transmitter can bind to the receptor and change the structure of the receptor, that leads the ion channel shut down or opened. The permeability of the membrane to ion will be changed at this time. Meanwhile, the chemical signal will be exchanged as electric signal (depolarization), and the electric signal (electric excitation) will be transferred to postsynapse cell …… For example, acetylcholine receptor exists as three structures, they can be opened for 1/1,000,000 second when acetylcholine molecule bind to them. Ion-channel-linked receptors can be sorted as positive ion channels (receptors for acetylcholine, glutamic acid, and 5-TH) and negative channels (receptors for glycine and γ－aminobutyric acid).

19. Ion-channel-linked receptor

20. A model structure of the receptor of acetylcholine

21. Ion-channel-linked receptor at the junction of nerve and muscle

22. G-protein-linked receptor: Trimeric GTP-binding regulatory protein is called as G protein located on plasma side of membrane. G protein is composed of subunit α, β, and γ. G protein is a switch during the signal transduction that can shut down when subunit α binds to GDP, opened up when subunit α binds to GTP. Subunit α is of GTPase activity that can hydrolyze GTP. G-protein-linked receptor is seven-times-transmembrane protein. The extracellular part of the receptor recognizes and bind signal molecule, and intracellular part of the receptor links to G protein. The extracellular signal bind to the receptor can cause the second messenger formed inside cell by the linked G protein. The receptors for neuron transmitters, peptide hormones are the G-protein-linked receptor. The receptors for the physical and chemical excitations of taste sense and visual sense are G-protein-linked receptors too. The signal ways mediated by G-protein-linked receptor include cAMP signal way and phosphatidylinositol signal way.

23. The molecule switch of G protein

24. G-protein-linked receptor is seven-times-transmembrane protein

25. Three structures of the acetylcholine receptor

26. cAMP signal way: Extracellular signal binds to the extracellular part of the G-protein-linked receptor. By the mediation of G protein, the intracellular part of the receptor will form the second messenger, cAMP signal that is intracellular signal. Components of cAMP signal: ①.    Activating hormone receptor (Rs) / inhibiting hormone receptor (Ri). ②.    Activating regulatory protein (Gs) / inhibiting regulatory protein (Gi). ③.    Adenylyl cyclase: A glycoprotein (150KD) that passes through the plasma membrane 12 times. With Mg2+ or Mn2+, adenylyl cyclase catalyze ATP into cAMP.

27. Mg2+ or Mn2+ Adenylyl cyclase

28. ④.    Protein Kinase A (PKA): PKA is composed of two catalytic subunits and two regulatory subunits. cAMP binds to regulatory subunits and releases out the activated catalytic subunits that can phosphate the serine and threonine residues of some proteins in cells to change the activity of them. ⑤.    cAMP phosphodiesterase: It can degenerate cAMP to form 5’-AMP, that stops signal.

29. Protein Kinase A

30. Degeneration of cAMP

31. The model of Gs regulation: When Gs is inactivated, α subunit is combined with GDP, and adenylyl cyclase has no activity. When the ligand, such as hormone, combines to Rs (extracellular part of receptor), the structure of receptor (Rs) will be changed and the Gs binding site will be explored and the ligand-receptor complex will bind to Gs. The structure of the Gs α subunit will be changed. This α subunit rejects GDP and combine GTP to activate it. Gs will releases out its α, β, and γ subunits, and explore the adenylyl cyclase binding site on α subunit. The explored and GTP combined α subunit will combine adenylyl cyclase and activate it. The activated adenylyl cyclase can cause ATP changed to cAMP. With the GTP hydrolysis, α subunit will return to original structure and be separated from adenylyl cyclase. The activation of adenylyl cyclase will be stopped. α subunit combine β and γ subunits. Gs regulation return back to original. The cholera enterotoxin can catalyze ADP bind to the α subunit of Gs, that cause α subunit to lose its GTPase activity resulting in GTP consistent combination to the α subunit. The α subunit will keep activated consistently, and the adenylyl cyclase will be activated forever. These pathological changes will lead the Na+ and water floated out of cells. The patient will take a severe diarrhea and dehydration.

32. The model of Gs regulation

33. The cAMP signal way can be summarized as the follows: Hormone G protein linked receptor G protein adenylyl cyclase cAMP cAMP dependent PKA Regulatory protein for gene transcription The different cell responses to cAMP signal way with different speed. For examples, The degeneration of glycogen to glycose-1-phosphate can be started within 1 second in muscle cells. But, it needs several hours in some secreting cells because activated PKA will enter nucleus to phosphorate CRE (cAMP response element) bound protein and regulate the expression of relative gene. CRE is the regulation region of DNA sequence to a gene.

34. cAMP signal and glycogen degeneration

35. cAMP signal and gene expression

36. The model of Gi regulation: Gi can inhibit adenylyl cyclase by the following routes: ① combine to the α subunit of adenylyl cyclase and inhibit the activity of enzyme. ② combine to the α subunit of free Gs by binding to the complex of β, γ subunits complex. As result, the activation of adenylyl cyclase by α subunit of Gs will be inhibited. The pertussis toxin can inhibit the binding of the α subunit of Gi to GTP, and block the inhibition of adenylyl cyclase by Ri receptor. So, the development and pathological syndrome of chin cough are associated with the inhibition of Gi regulation way. But, the detailed mechanism about this inhibition keeps unknown so far.

37. The model of Gi regulation

38. Phosphotidylinositol signal way: In the phosphotidylinositol signal way, extracellular signal molecules combine to the G protein linked receptor on membrane surface, and activate the phospholipase C (PLC-β) that hydrolyze 4,5-diphophotidylinositol (PIP2) into 1,4,5–triphophotidylinositol (inositol phosphate 3, IP3) and diacyl glycerol (DG) as two second messengers. Extracellular signals are exchanged as intracellular signals at this time. We call the signal system as double messenger system).

39. Phosphatidylinositol signal way

40. IP3 can combine to the IP3 ligand gate Ca2+ channel to open it, and the Ca2+ concentration in cell will be lifted up. The high Ca2+ concentration will activate every Ca2+ dependent protein. If you use Ca2+ carrier ionomycin to treat cultured cells, you will get same result as described above. DG can activate plasma membrane bound protein kinase C (PKC). PKC is distributed in cell plasma without activity usually, but when cell received some excitation, IP3 will make high Ca2+ concentration, then, PKC is translocated onto plasma membrane inside surface to be activated by DG. The activated PKC can phosphorate Ser/Thr residues of proteins causing different cell exhibited with different response. For examples, secretion of cell, contraction of muscle, proliferation and differentiation of cell. The role of DG can be mimicked by phorbol ester.

41. The roles of IP3 and DG

42. Ca2+ signal level can be regulated by calmodulin (CaM). Ca2+ bound CaM can activate CaM-Kinase. So, the response of cell to Ca2+ is depended upon the Ca2+ bound proteins and CaM-Kinases in cell. For example, CaM-Kinase II is adjacent at the synapse of neuron that is associated with memory formation. IP3 signal will be dephosphorylized or phosphorylized as IP2 or IP4 to be stopped. Ca2+ will be exported out from cell by the pumps of Ca2+ and Na+-Ca2+. DG will be stopped by two ways: 1. DG is phosphorylized as phosphatidic acid. 2. DG is hydrolyzed as monoesterglycerol.

43. The cancellation of Ca2+ signal

44. Other G protein linked receptors: 1．The G protein in chemical receptor: The gas molecules can combined to the G protein linked receptor in the chemical receptor and activate adenylate cyclase to form cAMP, and open cAMP-gated cation channel to depolarize the membrane to cause the neuron excitation. This excitation causes olfaction or taste sense. 2．The G protein in optic receptor: Rhodopsin (Rh) is the G protein linked receptor in optic receptor complex. Light can change the structure of Rh and degenerate Rh as retinene and opsin. The opsin can activate G protein. The activated G protein can activate cGMP phosphodiesterase to hydrolyze cGMP in retinal rod cells. The Na+ channel will be shut down and the retinal rod cells will be hyperpolarized, that causes visual sense. The serial changes described as above can be shown as the follows briefly: Light signal Rh activated G protein activated cGMP phosphodiesterase activated The level of cGMP decreased Na+ channel shut down Concentration of Na+ in retinal rod cell fallen down Retinal membrane hyperpolarized The secretion of neuron transmitters inhibited visual sense

45. Retinal rod cell G protein linked receptor G protein in optic receptor

46. Enzyme linked receptor: Enzyme linked receptor can be sorted as two types: 1. The receptors are of kinase activity, such as, peptide growth factors (EGF，PDGF，CSF) receptors. 2. The receptors are not of kinase activity, and they can link to non-receptor tyrosine kinase. For example, the super receptor family of cytokine. The receptors above can be activated when they combine to their ligands by a dimerization way. Six types of enzyme linked receptor were identified so far (Because they have kinase activity, we call them as receptor kinase): ① Receptor tyrosine kinase. ② Receptor linked tyrosine kinase. ③ Receptor tyrosine lipase. ④ Receptor Ser/Thr kinase. ⑤ Receptor guanylate cyclase. ⑥ Receptor linked histidine kinase. Receptor tyrosine kinase: 1. Tyrosine kinase: Tyrosine kinase can be sorted as three types: ① Receptor tyrosine kinase (it is located in membrane as a transmembrane protein). More than 50 types have been found in vertebrates. ② Plasma tyrosine kinase, such as, Src family, Tec family, ZAP70 family, and JAK family. ③ Nucleus tyrosine kinase, such as Abl and Wee. The extracellular part of receptor tyrosine kinase is to be bound by ligands (polypeptide or hormones). Intracellular part is catalytic domain. These receptors include EGF、PDGF、FGF and others.

47. The dimerization and self-phosphorylation of receptor tyrosine kinase

48. Receptor tyrosine kinases

49. The recognition domain between signal molecules: A 50 – 100 mer domains in signal molecules are homologous each other. These domains can mediate the signal recognition or be linked together to form the signal transduction pathways like computer connections to connect each parts as “Signal transduction network”. The domains include: SH2 domain (Src Homology 2 domain): 100mer. Mediate the combination of signal and the proteins that contain phosphate tyrosine. SH3 domain (Src Homology 3 domain): 50~100mer. Mediate the combination of signal and the proteins that contain prolines. PH domain (Pleckstrin Homology domain): 100~120mer. Combine to membrane surface phospholipids (PIP2, PIP3, IP3) to translocate the PH domain protein to membrane from plasma.

50. Ras signal pathway: Receptor tyrosine kinase (RTK) will be activated after signal combined, structure dimerized, and molecule phosphorated by itself. The activated RTK can activate Ras. Ras is a proto-oncogene family associated with cancer development. Raf is another proto-oncogene family. It is Sr/Thr protein kinase and also called as mitogen-activated protein kinase (MAPKKK). The N terminal of Raf can bind to Ras to be activated. Activated Raf will cause a serial reactions of protein kinase phosphorylation to take effects on the growth and differentiation of cells. The statement above is important to understand tumor development. RTK-Ras signal pathway (a serial of activations) can be described briefly as the follows: Ligand RTK adaptor GEF Ras Raf（MAPKKK） MAPKK MAPK Activated MAPK enters nucleus Transcription factors (Elk-1and others) Enhance proto-oncogenes (c-fos, c-jun) expression Tumors.