Topic 3 Cell Signalling

1. Topic 3Cell Signalling

2. Messengers • Chemical • Endocrine • Paracrine • Autocrine • Electrical

3. Endocrine Signals • Act over long distances • Distributed via the bloodstream • Examples include • Insulin • Peptide hormone—Islets of Langerhans—pancreas • Signals muscle and adipose to uptake glucose in response to high blood glucose • Nobel Prize—1923—Banting and Macleod—discovery of insulin

4. Paracrine & Autocrine Signals • Paracrine: • Act over short distances • Released locally • Example • Epidermal growth factor • Stimulates growth and development • Autocrine • Act on the cell that secretes them

7. Flow of Information • Chemical messenger=ligand • Ligand acts as primary messenger • Activation of other molecules • Secondary messenger system

8. Receptors • Usually transmembrane proteins • Exception is steroid hormone receptors • Ligand binding domain • Specific to signal that they receive • Specificity is via ligand binding domain • Can have different affinities for different ligands • Ligand binding affects intracellular activities by • Changing conformation of the receptor • Clustering of receptors

10. Two types of receptors G-protein coupled receptors Protein kinase linked receptors

11. GPCR structure • Single polypeptide • Main function—activation of G protein • 7 transmembrane domains • Ligands bind near extracellular domain • Several cytoplasmic domains near TM5, TM6, TM7 and maybe TM4 mediate G protein binding • Large protein superfamily

12. Structure of GPCR

13. G proteins • Bound to the inner side of the plasma membrane • 3 subunits • α, βand γ • Inactive form • 3 subunits associated with each other; α is also bound to GDP • Activated • GDP replaced by GTP; α subunit + GTP dissociate from β and γ subunits

14. Activation of GPCR http://www.nature.com/scitable/content/ne0000/ne0000/ne0000/ne0000/14673543/U4.cp2.1_nature01307-f1.2.jpg

16. Activated G Protein • Can activate ion channels • Activate 2nd messenger cascades • Cyclic AMP • Cyclic GMP • Phospholipase C pathway

17. Inactivation of G protein α subunit of G protein contains a GTPase GTP degraded to GDP α subunit reassociates with β and γ subunits

18. Cyclic AMP cascade http://t3.gstatic.com/images?q=tbn:ANd9GcQev_GY0IC6y5BsuY4GoQ-8UaWogpktHlZpStl29YimNMDMXGP7 • G protein activates adenylyl cyclase • Catalyzes conversion of ATP to cyclic AMP • Cyclic AMP activates protein kinases • Protein kinases phosphorylate other proteins • Activation of ion channels • Metabolic changes • Changes in transcription factors

20. Cyclic AMP http://psychology.jrank.org/article_images/psychology.jrank.org/neurotransmitters-and-neuromodulators.9.jpg

21. Termination of cAMP activity • Cyclic AMP activity terminated by a phosphodiesterase • Converts cyclic AMP to 5’AMP

22. cAMP activation of protein kinases

23. Cyclic GMP cascade http://t2.gstatic.com/images?q=tbn:ANd9GcQBJuFA6x0bdMfdJ27IcwR_oWPfDzTSaawOQ8mzeR13LVmColSLXQ Very similar to cyclic AMP Utilized guanidine instead of adenosine GPCRs activate guanylyl cyclase Activation of cGMP-dependent protein kinases

24. Phospholipase C pathways • Common membrane protein: • Phosphotidylinositol 4,5-biphosphate (PIP2) • ligands bind GPCR • G proteins activated • α subunit activates a membrane-bound enzyme • Phospholipase C (PLC) • PLC catalyzes hydrolysis of PIP2 into inositol triphosphate (IP3) and diacylglycerol (DAG)

26. Phospholipase C cleavage of PIP2 http://www.ncbi.nlm.nih.gov/bookshelf/picrender.fcgi?book=dbio&part=A1401&blobname=ch7f28.jpg

27. IP3 pathway IP3 was the polar head of the phospholipid Freely diffuses into cytoplasm Releases Ca2+ from intracellular stores

28. Phospholipase C http://psychology.jrank.org/article_images/psychology.jrank.org/neurotransmitters-and-neuromodulators.11.jpg

29. DAG pathway Nonpolar component of phospholipid Diffuses through the membrane bilayer Activates protein kinase C (PKC) Results in protein phosphorylaton

30. Ca2+ and PKC • Ca2+ released by IP3 can enhance activation of PKC • Activating calmodulin • Calmodulin + Ca2+ • Activation of calcium/calmodulin-dependent protein kinases

31. Disruption of GPCR Signalling Cascades • Vibrio cholerae • Intestinal tract • Bordetella pertussis • lungs

32. Calcium’s Role • Major role in regulation of cell functions • Maintained at low concentration in cytosol • Calcium ATPases • In neurons • Influx of extracellular calcium • Release of neurotransmitters • In cardiac and skeletal muscle • Released from sacroplasmic reticulum • Involved in contraction

33. Calcium Regulation in the Cell

34. Other roles • Fertilization • Ca release from spermsperm activation binding to mature eggfertilization • Release of internal stores of Ca in egg • stimulation of cortical vesicles to prevent polyspermy • Resumption of metabolic processes • Initiation of embryogenesis

35. Ca binding to effector proteins • Calcium can bind directly to some proteins altering their activity • Example is Calmodulin • Conformational change when Ca binds • Ca-Calmodulin can bind to protein kinases or phosphatases

37. βγ subunits of G protein Can activate protein receptors i.e.—muscarinic acetylcholine receptor

38. Protein Kinase-Associated Receptors • Binding of ligand to PK-associated receptor stimulates kinase activity • Transmission of signal via phosphorylation cascades • 2 major classes • Tyrosine kinase receptors • Serine-threonine receptors

39. Growth factors • Cell culture studies • Plasma—no cell growth • Whole blood with unactivated platelets but no red or white blood cells • Serum---cells grow • Clear fluid remaining after blood clots • During clotting, platelets release platelet-derived growth factor • Stimulates fibroblast growthscar

40. Growth factors • Receptor is tyrosine kinase • Other GFs include • Insulin • Insulin-like growth factor • Fibroblast growth factor • Epidermal growth factor • Nerve growth factor

41. Receptor Tyrosine Kinases • Single peptide chain • 1 transmembrane domain • Extracellular domain=ligand receptor • Cytosolic domain=protein kinase • Contains tyrosines that are targets for their own kinase activity • 1st discovered = Src • Encoded by src gene of avian sarcoma virus

42. Activation of RTKs • Ligand binds • RTKs aggregate • Tyrosine kinase phosphorylates tyrosines of neighbouring RTKs =autophosphorylation

43. Ras pathway • Involves a number of proteins including: • Ras – small monomeric G protein • GEF – guanine-nucleotide exchange factor • Sos—GEF that activates Ras • GRB2 – contains SH2 domain which binds to RTK

45. Ras • RTK phosphorylated • GRB2 and Sos form a complex • Bind to RTK activating Sos • Sos activates Ras to release GDP and bind GTP • Activated Ras phosphorylates threonine and serines of MEK (also known as MAPK) • MAPK phosphorylate transcription factors • GTPase activation protein—hydrolyzes GTP on Ras  Ras inactivation

46. RTKs activate other pathways • Phospholipase C pathway • RTK activates PLC-gamma • PLC-gamma has a SH2 domain • GPCR activates PLC-beta

47. Scaffolding • Formation of multiprotein complexes • Cascading is more efficient • Cellular responses restricted to a smaller area

48. Serine-ThreonineKinases • Receptor for transforming growth factor β • TGFβ binds to ser/thr kinase receptor • 2 types—type I and type II • Type II phosphorylates type I • Type I phosphorylates R-Smad • R-Smad complexes with Smad 4 • Moves to nucleus • Regulates gene expression

50. You should be able to: Compare and contrast GPCR and protein kinase receptors Describe the cAMP, cGMP, PLC, Smad and Ras pathways