1. SIGNAL TRANSMISSION WITHIN THE CELL Nela Pavlíková nela.pavlikova@lf3.cuni.cz

2. G-PROTEIN COUPLED RECEPTOR - 7 transmembrane segments

3. G-protein  heterotrimer, composed of 3 different subunits:    subunit ~ 33-55kD - binding place for GDP / GTP - intrinsic hydrolytic activity for GTP (it binds GTP = activation → hydrolyzing of GTP to GDP = deactivation) - in deactivated state - it has bound GDP molecule - it is associated with G  complex   subunit ~ 35kD   subunit ~ 15kD - creates G  complex  mammals: 20 different G proteins (each contains unique G  subunit + one of 5  subunits and one of 12  subunits)

5. GiGi GsGs GqGq

6. GsGs  stimulates adenylyl cyclase → synthesis of cAMP → activation of protein kinase A (PAK) → phosphorylation of nearby substrates  cholera toxin: it keeps G  s permanently activated → ↑cAMP G  i  after activation it inhibits adenylyl cyclase → ↓cAMP  pertussis toxin: G  i subunit unable to release GDP → incapable of activation → cannot inhibit adenylyl cyclase cAMP is involved in regulation of many ion channels

7. GqGq  not influenced by cholera toxin nor pertussis toxin  phospholipase C  → inositol-3-phosphate (IP 3 ) → ↑Ca 2+ in cell → CaMKII → diacylglycerol (DAG) → protein kinase C  both pathways lead to phosphorylation of nearby proteins

9. GtGt  „transducin“  sensitive to pertussis toxin → incapability to release GDP → inhibition of signalization  Vision: t 1 – black-and -white t 2 – colors  occurrence: retina, some stem cells  effector: phosphodiesterase-6 (PDE 6 ) → degradation of cGMP

10. GoGo  „other“  effectors not known  sensitive to pertussis toxin → incapability to release GDP → inhibition of signalization  occurrence: neural and endocrine tissues, mitotic spindle  early development: effect of serotonin on neuron migration

11. G  12/13  activation of REF („Rho guanine-nucleotide exchange factors“) → activation of Rho proteins  occurrence: ubiquitous

13. RECEPTOR TYROSINE KINASE

14.  growth factors, cytokines, hormones  17 subfamilies of receptors  dimerization of receptors necessary (even tetramers)  only one transmembrane segment (N-end extracellular, C-end intracellular)  tyrosine phosphorylation = creation of binding domains for cytosolic signaling proteins → binding → activation of signaling pathways  cytosolic signaling proteins: e.g. Src, phospholipase C , PI3-kinase  domains for binding on phosphorylated tyrosines: SH 2 domain (src), PTB domain (PI3-kinase)

15. RECEPTOR TYROSINE KINASE  endocytosis of activated receptor:  destruction of receptor in lysosome ← binding of ubiquitin molecule on kinase  endocytosed receptor signalization – e.g. NGF

17. RECEPTOR TYROSINE KINASE  Ras, Rho- monomeric GTPases - anchored in the inner membrane - a part of receptor tyrosine kinase signaling pathway - activation: GEF (guanine nucleotide exchange factor) - inhibition: GAP (GTPase activating factor) ↓ resistance → cancer  Ras → MAP kinase → signalization into nucleus (→ e.g. cell proliferation)  Rho → connect RTK with cytoskeleton (→ cell shape, motility, adhesion)

18. RECEPTOR TYROSINE KINASE

20.  PI-3 kinase - Akt

21. RECEPTORS ASOCIATED WITH TYROSENE KINASE  JAK-STAT

22. RECEPTOR SERINE/THREONINE KINASE  ligand: e.g. TGF-   SmaDs → regulatory protein  endocytosis → activation → degradation

23. NUCLEAR RECEPTORS  their ligands can cross the cytoplasmic membrane on their own  = transcription factors (in activated form they start transcription of target genes)  nuclear receptors activated by ligand vs. „orphan“ receptors  type I – receptor in cytosol → ligand binding → homodimerization → transport into nucleus → binding on „response element“ part of DNA → start of transcription  type II – receptor as heterodimer with RXR (and some corepressors) bound in nucleus on „response element“ → ligand into nucleus→ binds to receptor → dissociation of corepressors → start of transcription

27. answer on the question from the last seminar: synthesis of thyroid hormones