Cellular Receptors

1. Cellular Receptors Chapter 2

2. Binding of Drugs in/to Cells • Receptor = Drug “target” • Membrane protein • Enzyme • Nucleic acid • Most drugs bind receptors by weak, noncovalent forces (what are these?) • May be reversed by pH change

3. Molecular Recognition  Specificity • Cellular specificity • Not all receptors in all cells, tissues • Receptors selectively bind partic ligands • Stereoselectivity • No drug completely specific

5. Ligand/Receptor Interactions • Reversible, bimolecular reaction • D + R  DR  DR*  Response • Where R*=Receptor w/ conform’n change • Each will have rate constant • What does this remind you of??

6. Activating Drugs = Agonists • Drug/receptor binding •  conform’l change in receptor •  act’n “downstream” cell biochem pathway(s) •  tissue response • May bind at separate site on receptor • “Allosteric modulators” • Increases response to natural agonist

8. Some definitions • Affinity = tendency to bind receptor • Specificity • Association/dissociation constant • Efficacy = tendency to activate receptor • Full agonists elicit max response • Partial agonists elicit submaximal response

9. Antagonists Bind Receptors… • BUT no activation occurs • No conform’l change in receptor, so no pathway response • May keep agonists from binding • Competitive • Book ex: curare blocks ACh from receptors of neuromuscular junction  inhib’n muscle depolarization  paralysis • Allosteric modulators may decrease natural agonist binding • Best antagonists have efficacy=0

11. Targets for Drug Action

12. Receptor Superfamilies • Ligand-gated ion channels • G protein-coupled receptors • Receptor tyrosine kinases • Nuclear hormone receptors

15. Ligand-Gated Ion Channels • Brain, periph NS, excitable tissues (heart), neuromuscular junction • Nicotinic cholinergic receptors (neuromusc) • GABA receptors (brain) • Glutamate receptors (brain) •  change membr potential  fast synaptic transmission • Complex prot’s w/ multiple subunits

16. Book Ex: Nicotinic Receptor • Number of subunits differs w/ tissue • Antagonists differ • Allows selective blockade neuromuscular junction • Multiple binding sites for Ach • Excitatory •  incr’d Na+/K+ permeability  incr’d depol’n  incr’d probability of action potential • Direct transduction (no biochem intermediates)

18. Allosteric modulators may increase/decrease transmitter response in ligand-gated channels • Ex: benzodiazepines • Antianxiety; sleep disorders • Bind GABA ligand-gated receptors • GABA inhibitory • Increases ability of GABA to open channels

19. G Protein Coupled Receptors • Single subunit • 7 helices span bilayer • Agonists may bind extracell N-terminal domain, or between helices • Few allosteric modulators known • Cytoplasmic loop couples to G protein

21. G Proteins • Intermediary mol’s • Bind guanine nucleotides • Extrinsic (periph) prot’s at inside bilayer • Anchored to membr by fa chain • Shuttle between receptor, target prot’s • 3 subunits • GTPase activity by a

22. “Resting state” G prot – trimer w/ GDP occupying site on a subunit • Agonist binding receptor  conform’l change w/in cytoplasmic domain •  Receptor acquires high affinity for G prot  binding G prot to receptor • GTP replaces GDP • bg duplex dissoc’s from a-GTP • Diffuse along membr • Assoc w/ enzymes, ion channels  act’n or deact’n

25. Term’n activity w/ hydrol Pi from GTP w/ GTPase activity of a subunit • Trimer reunites • Single agonist binding can activate sev G-prot mol’s for sev prod’s/act’n results •  Amplification

26. Sev types G prot’s • Interact w/ diff receptors • Control diff effectors • Gs stim’s enz adenylate cyclase, PLC, others • Gi inhibits ad cyclase, PLC, others • Agonist specificity

27. Cellular Responses • Amplification of signal through second messengers that activate kinases • cAMP • Phosphatidylinositol •  Control regulatory enz’s through covalent mod’n •  Large, varied cell responses • GPCRs also control • PLA  eicosanoid release • Ion channels  depol’n, transmitter release, contractility, etc.

29. Examples of GPCRs • Receptors for • ACh (muscarinic) • Neuropeptides • Ephinephrine • Muscle (3 types), liver, fat, epithelium, neurons

30. Receptor Tyrosine Kinases • Single transmembr a helix • Large extracell domain • Agonist binding site • Large intracell domain • Some incorporate tyr kinase activity • Cytokine receptors assoc w/ cytosolic kinases • Agonist binding  act’n  dimerization • Monomeric form inactive

31. Dimerized receptors autophosphorylate tyr residues • Phosphorylated tyr attracts, binds SH-2 domain protein • Src Homology • Conserved seq recognizes phosphotyrosine on receptor • Various SH2-domain prot’s allow selectivity for spec receptors • Some are enzymes • Kinases • Phospholipases

32. Some SH-2 Domain prot’s are couplers for other cell prot’s w/ phosphorylated receptors • Phosphorylation cascades • Impt to cell division, diff’n • Ex: Ras/Raf/MAP kinase pathway • Impt to cancers

34. SH-2 Domain prot’s as couplers – cont’d • Ex: Jak-Stat Pathway • Impt for cytokines, growth hormone, interferons • Cytosolic kinase phosphorylates receptor dimer • Various Jak’s  specificity • SH-2 domain prot’s (Stat’s) attracted, phosph’d, dimerize •  Nucleus  gene expression

38. Nuclear Hormone Receptors • Intracellular • Most in nucleus • Some cytoplasmic • Three domains: • Agonist binding domain at C-terminal • Transcriptional control domain • DNA binding domain • Highly conserved • “Zinc fingers”

40. Ligands lipophilic • Traverse lipid bilayer • Examples: • Steroid hormones • Thyroid hormones • Vitamin D • Retinoic acid • Impt to embryo dev’t

41. Agonist binding to receptor  conform’l change •  Dimerization of receptors • Dimers recognize specific base seq’s on DNA near genes • Hormone responsive elements • ~200 bp upstream from genes • Binding DNA may activate or repress gene transcr’n • So “ligand-activated transcr’n factors”

43. Other Targets of Drugs • Ion Channels • Ligands bind voltage (as well as ligand-gated) channels • Block channel • Affect gating • Activation GPCRs  phosph’n channel prot’s • Affect channel opening • Ex: opioids, b-adrenoreceptor agonists • Modulation intracell Ca+2, GTP, ATP • Channels may bind these mol’s • Ex: sulfonylureas act at ATP-gated K+ channels on pancreatic B-cells

45. Enzymes • Drug may be substrate analog • Competitive or irreversible inhibitor • False substrate • Appears as substrate, so taken up • Not useful as product • Ex: 5-FU blocks DNA synth • Prodrugs • Metabolism  active agent

46. Carrier molecules • Impt for transport across cell membr’s • Have recognition sites for natural mol • Examples: • Cocaine, antidepressants inhibit noradrenaline uptake • Amphetamine acts as false substrate • Loop diuretics affect Na+/K+/Cl- transporter in renal tubule • Cardiac glycosides inhibit Na+/K+ pump

47. Single Agonist May Have Complex Effects • Families of receptors for agonists • Ex: ACh receptors muscarinic, nicotinic • Further subtypes • Some receptors very specific • Some receptors bind similar ligands • Book ex: dopamine structurally sim to norepi, can stim b1-adrenergic receptors • Multiple receptor subtypes for one ligand can coexist in single cell

49. Regulation of Receptors • Drugs, agonists decrease sensitivity of receptors to ligands • Fast: desensitization, tachyphylaxis • Gradual: tolerance, refractoriness, drug resistance • Usually w/ continuous exposure • Sensitivity can be increased • Sensitization, desensitization can occur by ligand to same ligand or another

50. May be due to • Change in receptors • Phosphorylation – inhibits ability to interact w/ G proteins • Slow conform’l change • Exhaustion of mediators • Ex: amphetamines relase amines from nerve terminals; when endogenous amines depleted, drug doesn’t work