biochemistry生物化學西文chapter5-protein function,modulation and evolution gpcr

1、Biochemistry Lecture (Oct 18, 2012)Chapter 5-5 Protein Function, modulation and evolution: G-protein coupled receptors (GPCRs) Also called seven-transmembrane (7TM) Receptor Allow cells to sense their environment; Activate G-proteins and downstream proteins; Transducing messages of photons (vision),

2、 odorants (smell), tastants (taste), hormones, neurotransmitters (behavior and mood), etc; By Prof. Chang Zengyi (昌增益教授）G -proteinReceptorEpinephrine, a catecholamine prepares an animal to “fight or flight”.Nerve signals and hormones from the brain alert the body. The adrenal gland releases stress h

3、ormones into the bloodstream. Cells throughout the body sense that something is happening via their receptors and respond accordingly.Epinephrine is also produced in the brain!GPCR is a superfamily, with b2 adrenergic receptor (b2AR) being a model system Human genome encodes about 1,000 GPCRs; Being

4、 targets of approximately 30% of all modern medicinal drugs! Ligand binding to GPCR activate the G-proteins, which modulate further downstream proteins and the physiological states of the cells.G protein cycle for b2ARConformational rearrangementsof GPCRGs heterotrimerDissociation of a & bc subunits

5、Effector proteinsActivated adenylyl cyclaseCa2+channelsHydrolysis of GTPSecond messengersActivated GPCREpinephrine(adrenaline)GGGProlonged exposure to ligands desensitizes GPCR Dissociation of ligand Phosphorylation and binding of b-arrestinIt is now commonly accepted that GPCR function as homo-olig

6、omers in living cellsHistory of understanding the structure of G-protein coupled receptorExample of rhodopsinoligomeroligomericGPCRGPCRGPCRGPCRrhodopsinrhodopsinrhodopsinmonomericmonomerPark et al., 20041905196219762000Fotiadis et al., (2006) Structure of the rhodopsin dimer: a working model for G-p

7、rotein-coupled receptors,Current Opinion in Structural Biology, 16:252259Earl W. Sutherland, Jr.for his discoveries concerning the mechanisms of the action of hormones1971 Nobel Prize in Physiology or Medicine1994 Nobel Prize in Physiology or MedicineAlfred G. GilmanMartin Rodbellfor their discovery

8、 of G-proteins and the role of these proteins in signal transduction in cells2012 Nobel Prize in Chemistry Brian K. KobilkaRobert J. Lefkowitzfor studies of G-proteincoupled receptorsAquaporins form hydrophilic transmembrane water channelsOriginally isolated as a 28 kDa contaminant of the Rh blood g

9、roup antigen from red blood cells, abundant in kidney tubes.(Nobel, 2003(Nobel, 2003)The potassium channel in bacteria is a homotetramer membrane proteinThe structure explains the exact mechanism by which potassium channel selectivity occurs. The Nobel Prize in Chemistry 1988“For the determination o

10、f the three-dimensional structure of a photosynthetic reaction center.” Dr Johann DeisenhoferUniversity of Texas Southwestern Medical Center, Dallas, USAProfessor Robert HuberMax-Planck-Institutfr Biochemie,Martinsried, FRGDr Hartmut MichelMax-Planck-Institutfr Biophysik,Frankfurt/Main, FRG.Structur

11、e of thephotosynthetic reaction center in purple Bacteria.A electron pathwas proposed.e-e-e-Periplasmic sideCytoplasmic sideCyt c2Cyt bc1complexMLCH The hormone epinephrine was found to act through the second messenger cAMP in activating glycogen degradation in liver cells (Sutherland, 1958) A heat

12、stable factor (cAMP) is produced in the presence of epinephrine and glucagon in liver homogenate. This factor then stimulates the formation of liver phosphorylase (rate-limiting for glycogenolysis in a fraction of the homogenate where the hormones are not present. EpinephrineCyclic AMPProteins inter

13、acting with the GTP (not ATP) found to needed to activate adenylyl cyclase (Gilman and Rodbell, 1960s).Concept of signal transduction proposed as a means of coupling information between signal-activated receptor (discriminator) and regulation of adenylate cyclase (signal amplifier); G-protein to be

14、the transducer.Radio-ligands or agonists used to detect GPCR receptors on specific cell membrane preparations (1970s, Lefkowitz) Radioactive iodine (125I) labeled adrenocorticotropic hormone (ACTH) specifically binds to adrenal extract and stimulates adenylyl cyclase. Similar radiolabeled (3H) -Adre

15、nergic Receptors agonists were used to characterize the epinephrine receptors in activating adenylyl cyclase activity.Lefkowitz et al (1970) ACTH receptors in the adrenal: specific binding of ACTH-125I and its relation to adenylyl cyclase. PNAS, 65:745-752. Lefkowitz et al (1974) Stereospecific (3H)

16、(minus) -alprenolol binding sites, beta -adrenergic receptors and adenylate cyclase. BBRC, 60:703-709. Coupling between ligand and G-protein binding events observed (1980, Lefkowitz)A ternary complex model, involving the extracellular ligand (agonist), transmembrane GPCR and intracellular G-protein,

17、 was proposed to serve as the active signalling unit.Allosteric coupling being mutual: binding of agonist increases the affinity for the G protein and vice versa.Limbird LE, Gill DM, Lefkowitz RL (1980) Agonist-promoted coupling of the beta-adrenergic receptor with the guanine nucleotide regulatory

18、protein of the adenylate cyclase system. PNAS,77:775. De Lean A, Stadel JM, Lefkowitz RL (1980) A ternary complex model explains the agonist -specific binding properties and the adenylate cyclase-coupled beta -adrenergic receptor. JBC, 255:7108. G protein and receptor aredifferent entities!The recep

19、tors purified and reconstituted into phospholipid vesicle Affinity chromatography using specific ligand-conjugated resins: b2-adrenoceptor, being a single polypeptide chain 60 kDa.A hormone-responsive adenylate cyclase system reconstituted from purified receptor and Gs and the catalytic moiety of ad

20、enylyl cyclase.Stimulated by agonists but inhibited by antagonists.Molecular details for the signaling process could be examined!Caron MG, Srinivasan Y, Pitha J, Kociolek K, Lefkowitz RJ (1979) Affinity chromatography of the beta-adrenergic receptor. JBC, 254:2923 -2927. Cerione, R.A.et al. (1984 )

21、Reconstitution of a hormone-sensitiveadenylate cyclase system. The pure beta-adrenergic receptor and guanine nucleotide regulatory protein confer hormone responsiveness on the resolved catalytic unit. JBC. 259, 9979 9982Genes encoding epinephrine receptor clonedHomology with rhodopsin revealed.Seven

22、 transmembrane helices predicted.Dixon RA et al (1986) Cloning of the gene and cDNA for mammalian beta-adrenergic receptor: primary structure and membrane topology. Nature 321:75 -79. RhodopsinEpinephrine receptorMembrane-spanning a-helices can be predicted from amino acid sequences via hydropathy p

23、lottingHydropathy index is calculatedFor successive segments (windows) of a given size, from 7 to 20 residues;For a window of 7 residues, the indices for residues 1 to 7, 2 to 8, 3 to 9, and so on, are plotted for the middle residue in each Window residue 4 for residues 1 to 7, etc);A region with about 20 residues of high hydropathy index ispresumed to be a transmembrane segment.Each amino acid is assigned a relative hydrophobicity indexStructure of the b2 adrenergicrecep