Enzyme linked receptors

Enzyme Linked Receptors, types and their mechanism of activation.

1. Enzyme linked receptors By: Faraza Javed Sidra Arif

2. Cell surface receptor Cell surface receptors are specialized integral membrane proteins that take part in communication between cell and outside world. Extracellular signaling molecules (usually hormones, neurotransmitters, cytokines, growth factors ) attach to receptor, triggering changes in function of cell. This process is called signal transduction.The binding initiates a chemical change on intracellular side of membrane.

3. Types Based on structural and functional similarities, membrane receptors are mainly divided into 3 classes: 1) Ion channel-linked receptor; 2) Enzyme-linked receptor 3) G protein-coupled receptor

4. Enzyme-linked receptors A group of transmembrane proteins that contain either intrinsic enzyme activity on their intracellular domain or associate directly with an intracellular enzyme. Upon ligand binding a conformational change is transmitted via a transmembrane helix which activates the enzyme, initiating signaling cascades

5. Basic receptor structure 1. Ligand -binding domain Extracellular to allow easy access for ligands . Strong affinity for specific ligands - allows different ligands that bind to same receptor to evoke particular cellular responses.   2. Transmembrane domain Contains a series of hydrophobic amino acids. Tethers receptor to cell membrane.

6. 3. Cytosolic "active" enzyme domain The intracellular domain of receptor itself is an enzyme or interacts directly with an enzyme.

7. Types 1. Receptor tyrosine kinases - phosphorylate specific Tyrosine residues on a small set of intracellular signaling molecules 2. Tyrosine- kinase -associated receptors - couple to proteins that have Tyrosine kinase activity 3. Receptor serine/ threonine kinases , which phosphorylate Serine or Threonine residues

8. 4. Histidine - kinase -associated receptors 5. Receptor tyrosine phosphatases - remove phosphate groups from Tyrosine 6. Receptor guanylyl cyclases - catalyze production of cGMP

9. 1)Receptor tyrosine kinases The predominant type of enzyme-linked receptors is family of the tyrosine kinase receptors , also referred to as receptor tyrosine kinases These receptors have intrinsic kinase activity encoded by intracellular domain, also referred to as the cytoplasmic tail

10. Mechanism of activation 1. Ligand binding to RTK monomers results in dimer formation . 2. Within the dimer the conformation is changed, locking the kinase into an active state . 3. The kinase of one receptor then phosphorylates a tyrosine residue contained in the "activation lip" of the second receptor.

11. 4. This forces the activation lip out of the kinase active site, allowing  ATP bind and resulting in enhanced kinase activity.This induces phosphorylation at further tyrosine residues . 5. Phosphotyrosine is a conserved "docking site" for many intracellular signal transduction proteins that contain SH2 domains .

12. a) Ras and the MAP Kinase Pathway When ligand binds to extracellular domain of growth factor receptor,a dimer is formed . Then tyrosine kinase domains phosphorylate tyrosine residues. This phosphorylation produces binding sites for proteins with SH2 domains. GRB2( Growth factor receptor-bound protein ) is one of these proteins. GRB2, with SOS bound to it, then binds to the receptor complex .

13. This causes activation of SOS. Son of Sevenless (SOS) refers to a set of genes encoding guanine nucleotide release factors for ras . When SOS is activated, it promote the exchange of GDP for GTP When ras has GTP bound to it, it becomes active. Activated ras then causes the activation of a cellular kinase called Raf-1.

14. Raf-1 kinase then phosphorylates another cellular kinase called MEK This cause activation of MEK. Activated MEK then phosphorylates another protein kinase called MAPK ( Mitogen -activated protein kinase ) causing its activation. This series of phosphylating activations is called a kinase cascade. It results in amplification of signal

15. Among the final targets of the kinase cascade are transcriptions factors( fos and jun ). Phosphorylation of these proteins causes them to become active and bind to the DNA, causing changes in gene transcription

16. Clinical significance of ras and map kinase pathway MAP kinase pathways regulate a wide range of physiological responses, including cell proliferation, apoptosis, cell differentiation, and tissue development. MAP kinase pathways are regulated by Ras proteins, which are found to be mutated and constitutively active in approximately 30% of all human cancers.

17. RAS and cancer The majority of mutations in ras decrease the intrinsic rate of GTP hydrolysis by ras and make the molecule significantly less sensitive to GTP hydrolysis. Thus, the outcome is a molecule that is predominantly GTP bound and therefore constitutively active .

18. It is now essentially independent of growth factor stimulation and continues to activate downstream pathways in the absence of any stimulation. Oncogenic ras is capable of transforming immortalized fibroblasts or epithelial cells

19. Raf and cancer Raf protein kinase are known to regulate proliferation, differentiation. Mutated Raf-1 is constitutively active and possesses in vitro transforming potential The potential for Raf-1 to play a broad role in tumorigenesis is evidenced by its ability to become activated by either PKC or the antiapoptotic protein Bcl-2 in a Ras -independent manner

20. Importantly, raf mutations have been identified in a range of human tumors. Therefore, the collective evidence suggests that Raf-1 is a viable anticancer drug target The first drug licensed to inhibit raf kinase is sorafenib . Other Raf inhibitors, encorafenib , dabrafenib , vemurafenib

21. b) Fibroblast Growth Factor The fibroblast growth factor family constitutes one of the most important groups of paracrine factors that act during development. They are responsible for determining certain cells to become mesoderm, for the production of blood vessels, for limb outgrowth, and for the growth and differentiation of numerous cell types.

22. Activation of the receptors Fibroblast growth factor binding to their receptors causes the receptors to dimerize , and this results in the activation of their protein tyrosine kinases . These kinases phosphorylate each other and initiate downstream signaling . There are three components of this signal. The main signal involves the activation of the ras protein and the MAP kinase cascade

23. In addition, the activated receptor stimulates phospholipase C to split PIP 2 into IP 3 and DAG. A third signal involves the phosphorylation of the Stat1 transcription factor and its subsequent translocation into the nucleus

24. Clinical significance Mutations in FGFR1 can cause the Pfeiffer syndrome , a malformation syndrome characterized by limb defects and by the premature fusion of the cranial sutures ( craniosynostosis ) that results in abnormal skull and facial shape There is also strong evidence from sequencing studies of candidate genes involved in clefting that mutations in the FGFR1 gene may be associated in the pathogenesis of cleft lip and/or palate

25. On a molecular level, mutations that affect FGFR2 are associated with marked changes in osteoblast proliferation and differentiation. Alteration in FGFR2 signalling is thought to underlie the craniosynostosis syndromes

26. There are two mechanisms of altered FGFR2 signalling . The first is associated with constitutive activation of FGFR, where the FGFR2 receptor is always signalling , regardless of the amount of FGF ligand . This mechanism is found in patients with Pfeiffer syndrome.  

27. The second, which is associated with Apert syndrome (malformations of the skull, face, hands and feet) is a loss of specificity of the FGFR2 isoform , resulting in the receptor binding to FGFs that it does not normally bind

28. c) insulin Mediate their activity by causing the addition of a phosphate group to particular tyrosines on certain proteins within a cell. Insulin binds to the tyrosine kinase receptor, located on the cell membranes of the target cells on skeletal muscle tissue, fat tissue, and liver.  The "substrate" proteins that are phosphorylated by the Insulin Receptor include a protein called "IRS-1" for "insulin receptor substrate 1

29. IRS-1 binding and phosphorylation eventually leads to an increase in the high affinity glucose transporter (Glut4) molecules on the outer membrane of insulin-responsive tissues, including muscle cells and adipose tissue, and therefore to an increase in the uptake of glucose from blood into these tissues

30. In other words, the glucose transporter Glut4 is transported from cellular vesicles to the cell surface, where it then can mediate the transport of glucose into the cell.

31. Effect of insulin on glucose uptake and metabolism Insulin binds to its receptor which in turn, starts many protein activation cascades. These include 1)translocation of Glut-4 transporter to the plasma membrane and influx of glucose 2)glycogen synthesis 3)Stimulates protein synthesis and inhibits proteolysis 4)Fatty acid synthesis

32. 2)Tyrosine- Kinase -associated Receptors Many cell-surface receptors depend on tyrosine phosphorylation for their activity and yet lack an obvious tyrosine kinase domain. These receptors act through cytoplasmic tyrosine kinases , which are associated with receptors and phosphorylate various target proteins when the receptors bind their ligand . The receptors thus function in much same way as receptor tyrosine kinases , except that their kinase domain is encoded by a separate gene and is noncovalently associated with receptor polypeptide chain.

33. The JAK/STAT Signaling Pathway Many cytokines, lymphokines , and growth factors signal through receptor tyrosine kinases that are associated with and activate Janus kinases (JAKs) Ligand -induced dimerization of the receptor induces the tyrosine phosphorylation of the associated JAKs, which, in turn, phosphorylates tyrosine residues on the cytoplasmic tail of the receptor.

34. These phosphorylated tyrosines serve as docking sites for SH 2 domain of the STAT(signal transducer and activator of transcription) protein, and JAK catalyzes the tyrosine phosphorylation of the receptor-bound STAT, Followed by translocation of the STAT dimer to the nucleus. STAT dimers bind to specific DNA response elements in the promoter region of target genes to activate gene expression .

35. a) IL-2 IL-2 receptors are expressed on T cells ,B cells,natural killer cells, monocytes , macrophages . IL-2 stimulation induces activation of Janus family tyrosine kinases JAK1 and JAK3,These kinases induce tyrosine phosphorylation of STATs (signal transducers and activators of transcription) and various other downstream targets

36. The downstream signaling pathways activated by IL-2 also involves mitogen -activated protein kinase and phosphoinositide 3-kinase signaling modules, leading to both mitogenic and anti-apoptotic signals. IL-2R signaling activates PI3K which catalyses phosphorylation of inositol phosphates. These act as second messengers and recruit molecules such as Akt kinase to the cell membrane

37. Akt kinase is further activated by phosphorylation and subsequently positively or negatively regulates the activity of downstream targets like PKB Proapoptotic proteins which can be phosphorylated and inhibited by PKB include BAD (BCL2 Antagonist of Cell Death) and forkhead family of transcription factors which regulate the genes responsible for determining whether activated T-Cells survive, proliferate, or die.

38. Clinical significance Differentiation of T lymphocyte The interaction of IL-2 with the IL-2 receptor induces proliferation and differentiation of a number of T lymphocyte subsets, and stimulates a cytokine cascade that includes various interleukins, interferons and tumour necrosis factors. Antitumour effects of IL-2 appear to be mediated by its effects on natural killer, lymphokine -activated killer (LAK) and other cytotoxic cells.

39. Type 1 diabetes IL-2 is necessary during T cell development in the thymus for maturation of regulatory T cells (T- regs ). After exiting from the thymus, T- Regs function to prevent other T cells from recognizing and reacting against self antigens, which could result in autoimmunity. T- Regs do so by preventing the responding cells from producing IL-2 .

40. Type 1 diabetes results from progressive immune-mediated destruction of pancreatic β-cells and associated metabolic dysfunction. Combined genetic and immunological studies now highlight deficiencies in both interleukin-2 (IL-2) receptor and its downstream signaling pathway as a central defect in the pathogenesis of type 1 diabetes

41. b) Interleukin 6 (IL-6) Acts as both a pro-inflammatory and an anti-inflammatory cytokine.IL-6 is secreted by T cells and macrophages to stimulate immune response. IL-6's role as an anti-inflammatory cytokine is mediated through its inhibitory effects on TNF-alpha and IL-1, and activation of IL-1ra and IL-10.

42. IL-6 signaling pathway and its regulation IL-6R is expressed on some cells, including immunocompetent cells and hepatocytes IL-6 signals through activation of JAK kinases and activation of ras -mediated signaling

43. Activated JAK kinases,STAT3 also upregulates the transcription of genes encoding the SOCS3 ( Suppressor of cytokine signaling 3 proteins)—intracellular negative-feedback factors that inhibit the JAK–STAT pathway.

44. Clinical significance Constitutive Activation of Jaks /Stats in Cancer In contrast to normal cells, in which Stat tyrosine phosphorylation occurs transiently, it has been determined that Stats 1, 3, and 5 are persistently tyrosine phosphorylated in most malignancies (particularly Stat3).

45. The mechanisms by which Stat3 is persistently or constitutively tyrosine phosphorylated in cancers include increased production of cytokines and cytokine receptors a decrease in the expression of the SOCS proteins loss of tyrosine phosphatases .

46. Rheumatoid arthritis IL-6 induces B-cell differentiation and has been shown to induce B-cell antibody production. Neutrophil migration from blood to tissue is a characteristic feature of inflammation

47. Upon entry, activated neutrophils release proteolytic enzymes and reactive oxygen intermediates resulting in tissue destruction and joint damage in RA. Neutrophils express membrane-bound IL-6R and are activated by IL-6. A humanized anti-IL-6R monoclonal antibody, tocilizumab (TCZ) is approved for arthritis

48. c) Erythropoietin Erythropoietins ( Epo ) is a hormone required for erythropoiesis . Epo exerts its function through the EpoR ( Epo Receptor). Following binding of Epo to its receptor, the receptor forms dimers and undergoes phosphorylation by physically associating and interacting with the tyrosine kinase JAK2 (Janus Kinase-2).

49. Once phosphorylated , these tyrosine residues allow the recruitment and activation of a number of downstream adaptors and effectors including STAT5 (Signal Transducers and Activators of Transcription factor-5), PI3K (Phosphoinositide-3 Kinase )

50. EPO-R in clinical disorders Polycythemia Polycythemia is clinical disorder characterized by an absolute increase in red blood cell mass and is associated with high serum EPO and intact EPO-R signaling. The extracellular domain of the EPO-R binds circulating EPO,and the intracellular domain controls signal transduction and has regulatory region that acts as a brake on red cell production.

51. Some cases of polycythemia were found to be associated with EPO-R mutations that were characterized by mutations with consequent truncations of regulatory region of the EPO-R. This region includes Tyr 429, which in its phosphorylated form is required for binding of SHP1, negative regulator of Tyr phosphorylation and signal transduction Loss of EPO-R SHP1 binding site prevents dephosphorylation of JAK2 and activation of STAT5, leading to hypersensitivity to EPO

52. 5)Receptor tyrosine phosphatases Receptor tyrosine phosphatases (RTP) are cell-surface proteins that dephosphorylate intracellular targets to activate signalling cascades. An important protein tyrosine phosphatase is CD45 protein which is found on surface of all white blood cells and has an essential role in activation of both T and B lymphocytes by foreign antigens

53. Key to ability of TCR to deliver intracellular signals is its interactions with protein tyrosine kinases CD45 activity is at very early steps of TCR signaling, indicating that CD45 is required for functional coupling of the TCR and its PTKs

54. Tenascin -C which is ligand of receptor protein-tyrosine phosphatase , expressed on the surface of certain glial cells in the mammalian brain. It binds to a receptor protein (called contactin ) on developing nerve cells indicates that their specific functional roles in stem cell neurobiology,which will be important for the therapeutic application of this new technology in facilitating nervous tissue repair and regeneration  

55. The findings show possibility that some receptor tyrosine kinases and receptor tyrosine phosphatases may collaborate when they bind their respective cell-surface-bound ligands —with the kinases adding more phosphates and the phosphatase removing fewer—to maximally stimulate tyrosine phosphorylation of selected intracellular signaling proteins

56. Reference John C, Torben J.Text book of receptor pharmacology(2 nd edition) pharmacologycorner.com/animation-showing-the- jak -stat- signalling -pathway-mechanism/ www.ncbi.nlm.nih.gov/books/NBK26822/ www.inkling.com/read/marks-medical-biochemistry-lieberman-marks-4th/chapter-11/iii--plasma-membrane-receptors