receptor tyrosine kinase-rtk signaling

Slide 1: 

Previously looked at the GPCR pathway How adenylyl cyclase and phospholipase C-? activations are mediated by G proteins Next we will be discussing the RTK pathway GPCR pathway RTK pathway Intracellular communication systems

Enzyme-linked receptors : 

Enzyme-linked receptors The RTK pathway is mediated by another class of receptors

Slide 3: 

PLC-?, an isozyme of PLC is activated through receptor tyrosine kinase (RTK) signaling pathway

Isozymes : 

Isozymes Enzymes that differ in amino acid sequence yet catalyze the same reaction

Slide 5: 

Like PLC-?, PLC-? catalyzes PIP2 ? IP3+DAG hydrolysis

Slide 6: 

PLC-? consists of domain X & Y PLC-? consists of domains X,Y, SH2 & SH3 Activation of PLC- ? does not occur through GPCRs but through RTK

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The two isozymes activate different signaling pathways because of the difference in amino acid sequence

Slide 8: 

Proteins with SH2 domains may interact with P-Tyr residues on activated RTKs

Slide 9: 

We will now look at the activation process of RTKs

Receptor tyrosine kinase is an example of an enzyme-linked receptor : 

Receptor tyrosine kinase is an example of an enzyme-linked receptor

Slide 11: 

There are seven receptor tyrosine kinase subfamilies (note : non-receptor tyrosine kinases or cytosolic tyrosine kinases also exist)

Some responses mediated by receptor tyrosine kinases : 

Some responses mediated by receptor tyrosine kinases

Slide 13: 

Insulin receptor is an example of a receptor tyrosine kinase Shown here is the insulin-bound receptor Note that the two intracellular tyrosine kinase domains within the receptor interact when insulin binds We will come back to the insulin receptor later on

Peperiksaan Pertengahan Semester II Sesi 2008-2009 : 

Peperiksaan Pertengahan Semester II Sesi 2008-2009 24 Februari 2009 8.30-10.00 mlm Dewan Gemilang

Slide 15: 

How do cells decide to divide or to differentiate? This is one question we hope to address in today’s lecture

Slide 16: 

The answer is related to how binding of certain extra-cellular signals to RTKs affect a downstream kinase mediating cell proliferation

Slide 17: 

Receptor Tyrosine Kinases - activation process dimerization autophosphorylation

Slide 18: 

Docking of these intracellular signaling proteins onto receptor P-Tyr residues occur via their SH2 domains

Slide 19: 

MAPK – Mitogen-activated Protein Kinase Receptor Tyrosine Kinases Function in Mitogenic (Growth Factor) Signaling GRB2 - growth factor receptor binding protein, Ras – monomeric G-protein

Slide 20: 

Ras activation occurs by GDP-GTP exchange In growth factor signaling, the GEF for Ras is Sos

Slide 21: 

This is Grb2-Sos in growth factor signaling (where Sos is the specific GEF for Ras)

Slide 22: 

Activation of Ras may occur through different pathways Other than RTKs, Ras may also be activated through GPCRs e.g. in brain tissues, Ras may be activated by : - Ca2+ & DAG - GEF that is not Sos

Slide 23: 

MAP kinase activation

MAP kinase activation involves a cascade : 

MAP kinase activation involves a cascade MAP kinase is activated by specific phosphorylations catalyzed by a kinase (i.e. MAP kinase kinase) In turn MAP kinase kinase is also activated by a kinase (i.e. MAP kinase kinase kinase!)

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MAP kinase activation Ras protein is an example of an upstream activator of the MAP kinase cascade

Slide 27: 

Summary- growth factor signaling

RTK-mediated responses : 

RTK-mediated responses EGF EGF receptor stimulates proliferation of various cell types

MAP kinase is translocted into the nucleus to influence cell proliferation : 

MAP kinase is translocted into the nucleus to influence cell proliferation MAPK-P transcription factor Elk-1?Elk-1-P Elk-1-P interacts with c-fos & c-jun genes ? transcription (protein products = Fos & Jun) activation of cell proliferation

Slide 37: 

In precursor neuronal cell lines, EGF causes MAP kinase to be active for a few minutes resulting in cell division (transient activation) NGF causes MAP kinase to be active for a few hours resulting in differentiation (sustained activation)

RTK-mediated responses : 

RTK-mediated responses Insulin insulin receptor stimulates carbohydrate utilization

Slide 40: 

Binding of insulin to its receptor activates a tyrosine kinase domain Activated tyrosine kinase then phosphorylates the insulin receptor substrate (IRS)

Slide 41: 

Fig. 12-6

IRS then acts as docking sitesfor SH2 proteins : 

IRS then acts as docking sitesfor SH2 proteins

Slide 43: 

P-Tyr on IRS Grb2 with SH2 domain & SH3 SH2 domains allow interactions of insulin signaling with other pathways PLC? with SH2 domain PI3K with SH2 domain

Slide 44: 

Of the pathways interacting with the IRS, we have discussed : the Ras-MAP kinase pathway the PLC? (inositol-phospholipid) pathway What we have not looked at is the PI3K pathway which is related to the effects of insulin on carbohydrate utilization

Pengaktifan IRS mempengaruhi pengisyaratan : 

Pengaktifan IRS mempengaruhi pengisyaratan PKB sebagai satu lagi kinase bergantung- pengutus kedua

How insulin stimulates carbohydrate utilization : 

How insulin stimulates carbohydrate utilization Activation of insulin receptor causes : Cellular glucose uptake rises due to an increase in plasma membrane glucose transporters (GLUT4) Glycogen synthase is maintained in an active form

Slide 47: 

Kesan-kesan perantaraan IRS Pengaktifan protein : i. PLC-? ? IP3 + DAG ii. PI3K ? PIP2 + PIP3 Kesan : IP3 ? Ca 2+ , DAG mengaktifkan PKC PIP2 mengaktifkan PKB (mengaruh pengambilan glukosa ke dalam sel, sintesis glikogen & sintesis protein)

About 30% of human cancers are related to ras mutations : 

About 30% of human cancers are related to ras mutations

Slide 49: 

Gen ras mutan menghasilkan produk protein Ras hiperaktif Pengaturan aktiviti Ras normal : GEF (mengaruh penukaran GDP?GTP) GAP (mengaruh aktiviti GTPase lemah @ Ras) Pengaturan aktiviti Ras hiperaktif : i. aktiviti GTPase tidak boleh diaruh GAP (rintang terhadap aruhan GTPase perantaraan GAP) ii. Ras hiperaktif sentiasa terikat-GTP & teraktif

Slide 50: 

Hiperactive Ras activity is not regulated by GAP ? Ras remains in the active state GTP Exchange Factor GTPase Activating Protein

Mutations or over-expression of tyrosine kinase receptors are also common in cancers (e.g. cervical & ovarian cancers) : 

Mutations or over-expression of tyrosine kinase receptors are also common in cancers (e.g. cervical & ovarian cancers)

Dalam sistem tumbuhan,kasked MAP kinase aktif apabila etilena tidak hadir : 

Dalam sistem tumbuhan,kasked MAP kinase aktif apabila etilena tidak hadir

Slide 55: 

Dalam sistem tumbuhan, etilen menyahaktifkan MAP kinase

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Termination of signaling may occur at various levels

Slide 58: 

Penamatan isyarat tapak jalan GPCR

Slide 59: 

Penamatan isyarat tapak jalan GPCR

Termination of GPCR-mediated signaling : 

Termination of GPCR-mediated signaling

Penamatan respons perantaraan cAMP : 

Penamatan respons perantaraan cAMP @ peringkat reseptor (nyahpekaan : pemfosfatan reseptor sewaktu ligan masih terikat & interaksi dengan arestin) @ peringkat protein G (GTPase intrinsik) @ peringkat enzim pelaksana iaitu penghasilan cAMP (fosfodiesterase) @ peringkat PKA (fosfatase)

Slide 62: 

Pemfosfatan reseptor 7-TMS menghalang pengaktifan protein G Domain sitoplasma reseptor teraktif difosfatkan oleh kinase reseptor berkait - protein G (GRK) Interaksi reseptor terfosfat dengan protein arestin menghalang interaksi dengan molekul protein G lain

Slide 63: 

Bagaimana GRK & arestin menghentikan respons G protein-linked receptor kinase (GRK) memfosfatkan reseptor Arrestin mengikat kepada reseptor terfosfat

Slide 64: 

Protein G mempunyai aktiviti GTPase intrinsik GTPase menghidrolisis GTP kepada GDP dan proteinG kembali ke bentuk tak aktif Reseptor juga boleh kembali ke bentuk tak aktif apabila tiada lagi isyarat ekstrasel / ligan mengikat Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings Fig. 11.7c

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Hidrolisis oleh GTPase menukarkan GTP kepada GDP pada G? dan menceraikannya daripada protein sasaran

Slide 66: 

Aktiviti GTPase protein G GTPase GTP GDP tindak balas hidrolisis intrinsik lemah diaruh oleh GAP (protein pengaktif GTPase) menceraikan protein G daripada enzim pelaksana & menghasilkan protein G tak aktif (menghentikan respons)

Slide 67: 

Pengaruh GAP ke atas hidrolisis GTP protein G GAP GAP GAP GAP mengaruh aktiviti GTPase

Slide 68: 

Many cell-surface ligand-receptor complexes are taken up into cells by a process called receptor mediated endocytosis Ligand binds receptor Ligand-receptor complex goes into “coated pit” Ligand-receptor complex is internalized by endocytosis into the cell (reduces #of receptors on cell surface). Ligand is often degraded in the lysosome The receptor itself is either degraded or recycled to the plasma membrane.

Slide 69: 

Penamatan respons cAMP oleh fosfodiesterase cAMP fosfodiesterase

Slide 70: 

adenilil siklase ATP cAMP PKA PKA tak aktif aktif protein protein-P (@ Ser / Thr) respons sel X fosfatase Penamatan respons oleh tindakan fosfatase

Similar with phospholipase C-? - mediated signaling : 

Similar with phospholipase C-? - mediated signaling

Slide 72: 

Penamatan respons perantaraan PLC- ? Penyahaktifan protein Gq GTPase dalaman G? GTP G? GDP Penguraian IP3 : IP3 + H2O IP2 Pengembalian [Ca 2+ ]sitosol kepada normal (10-7 M) melalui pam Ca 2+ @ membran plasma & retikulum endoplasma Hidrolisis DAG Nyahfosfatan oleh fosfatase

Slide 73: 

Molecular Biology of the Cell, 2002 Ca2+ dipam secara aktif dari sitosol ke luar sel Ca2+ dipam dari sitosol ke dalam ER dan mitokondria

Slide 74: 

Pengisyaratan perantaraan Ras ditamatkan melalui penyahaktifan Ras Penyahaktifan Ras berlaku melalui : i. tindakan GAP - mengaruh aktiviti GTPase @ Ras iaitu hidrolisis Ras (GTP)?Ras (GDP) ii. tindakan fosfatase – menyahfosfat residu fosfotirosin @ reseptor atau @ protein sasaran terfosfat tirosin

Slide 75: 

Penyahaktifan MAP kinase Penyahaktifan MAPK menamatkan pengisyaratan perantaraannya Penyahaktifan melibatkan pengeluaran kumpulan fosfat (nyahfosfatan) daripada residu P-Tyr / P-Thr oleh : fosfatase sedia ada (tanpa sintesis protein baru) fosfatase kespesifikan berganda (pengekspresan fosfatase ini perlu diaruh molekul isyarat ekstrasel & memerlukan masa untuk sintesis protein)

Slide 78: 

ADP ribosylation

Slide 82: 

Gangguan terhadap sistem pengisyaratan boleh menyebabkan penyakit

Slide 83: 

Mutasi reseptor berkait protein G & penyakit retinitis pigmentosa Keadaan normal : GRK reseptor rhodopsin (teraktif) reseptor-P penamatan pengisyaratan Keadaan berpenyakit : reseptor mutan tidak difosfatkan & pengisyaratan berpanjangan Akibat : kematian sel fotoreseptor & keadaan buta

Slide 84: 

Mutasi reseptor berkait protein G & penyakit diabetes bayi Keadaan normal : vasopresin + reseptor @ sel kidney penghasilan urin pekat Keadaan berpenyakit : pengaktifan reseptor mutan tidak menghasilkan urin pekat Akibat : dehidrasi, pertumbuhan perlahan, kecacatan mental, maut

Slide 85: 

Mutasi protein G? & penyakit ‘precocious puberty’ Keadaan normal : pengaktifan reseptor LH @ sel endokrin testis penghasilan testosteron Keadaan berpenyakit : protein G? mutan sentiasa aktif tanpa kehadiran ligan Akibat : terlalu cepat dewasa

Slide 86: 

Mutasi protein G? & penyakit ‘hipoparatiroidisme’ Keadaan normal : pengaktifan reseptor @ kelenjar paratiroid penghasilan hormon paratiroid Keadaan berpenyakit : protein G ? mutan sentiasa tak aktif & hormon paratiroid tidak terhasil Akibat : hipoparatiroidisme

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Toksin kolera mengakibatkan penghasilan cAMP berpanjangan Subunit ? toksin kolera memasuki sel epitelium intestin hos Protein G s? Protein G s? - ADP ribosil Protein G s? - ADP ribosil tidak boleh dihidrolisis oleh GTPase Adenilil siklase & penghasilan cAMP terangsang secara berlarutan gerak balas sel : rembesan air berlebihan

Tindakan toksin kolera : 

Tindakan toksin kolera Protein Gs pada sel epitelium hos mengikat kepada kumpulan ADP ribosil dengan kehadiran toksin kolera Tidak dikenali oleh GTPase & sentiasa aktif

Tapak jalan pengisyaratan adenilil siklase : 

Tapak jalan pengisyaratan adenilil siklase

Slide 91: 

Toksin pertussis (penyebab whooping cough) menyebabkan ADP-ribosilasi Gi Penambahan kumpulan ADP-ribosil @ protein Gi menyebabkan : i. tidak berlaku pertukaran GDP- GTP & pengaktifan protein G dihalang ii. interaksi Gi dengan adenilil siklase tidak berlaku

Slide 92: 

Perencatan Gs and Gi oleh toksin bakteria Toksin kolera : ADP ribosilasi residu Arg pada subunit ?s of Gs? perencatan aktiviti GTPase berkaitan Toksin pertussis : ADP ribosilasi residu Cys pada subunit ?i of Gi ? tidak boleh menghalang aktiviti adenilil siklase ?Kedua-dua toksin menyebabkan peningkatan aras cAMP © 2000 by W. H. Freeman and Company. All rights reserved.

Slide 93: 

Ca2+ bersama DAG mengaktifkan PKC

Slide 94: 

Trade Name: Viagra G-Protein Mediated Signaling Pathways Regulate Smooth Muscle Contraction

Slide 95: 

Coupling of signal transduction pathways: Protein phosphorylation and protein interaction domains. Adopted from Paul Shapiro