529235 Growth factors and their receptors, 3 ECTS credits Time: 16.3. - 5.5.2011, Wed and Thu at 14-16 Place: Viikki, Biocenter 2, Auditorium 1041 Organizers: Institute of Biotechnology and MBIOT Completion: Lectures, seminars Evaluation: Examination. Grading scale: 0-5 Responsible person: Prof. Mart Saarma Other lecturers: Kari Alitalo, Urmas Arumäe, Marja Mikkola, Pia Runeberg-Roos, Juha Partanen and Irma Thesleff
Growth Factors and their Receptors 529235, 3 ECTS credits Objective: The objective of this lecture series is to give the students a thorough view of the structure and function of the growth factors. It aims to describe the roles of growth factors in development and disease, and their use in biotechnology and biomedicine. Contents: The lecture course will deal with the structure and function of growth factors, analysis of signaling pathways, roles of growth factors in development, pathology and diseases. Growth factors functioning in different tissues and organs will be presented. Biotechnological and biomedical applications of growth factors will be discussed.
Program Wed 16.3. Discovery of growth factors. Basic principles of growth factor action. Mart Saarma Thu 17.3 Structure and synthesis of growth factors. Growth factorreceptor interactions. Signalling pathways. Mart Saarma Wed 23.3. Neurotrophic factors in development and disease. Mart Saarma Thu 24.3. Growth factors and cell death. Urmas Arumäe Wed 30.3. Integration of growth factor pathways in embryonic development. Irma Thesleff Thu 31.3. Cell biology of GDNF and its receptors. Pia Runeberg-Roos
Program Wed 6.4. FGFs and their receptors. Growth factors controlling development Juha Partanen Thu 7.4.. Transforming growth factors and tumor necrosis factors. Marja Mikkola Wed 13.4. WNT signalling in development and disease. Marja Mikkola Thu 14.4. 14.15 Receptors involved in the cell death. Urmas Arumäe 15.15 Growth factors, receptors and inherited diseases. Mart Saarma Easter week, no lectures Wed 27.4. Biotechnology and medical use of growth factors. Mart Saarma Thu 28.4. at 14-16 VEGF family of growth factors. Note place: Biomedicum 1, Haartmanin-katu 8, lecture room 1 (ground floor) Kari Alitalo Thu 5.5. Final Exam Note place INFO2
Wednesday 16.3.2011 Discovery of growth factors. Basic principles of growth factor action.
What are growth factors and how they act? Growth factors are secretory proteins that bind to the specific receptors on the cell surface and activate them Some of the growth factors are membrane bound and/or are activated upon cleavage Proteolytic cleavage
GDNF Family Ligands
Conserved Dopamine Neurotrophic Factor - CDNF and MANF form a new family that is conserved in evolution Lindholm et al. Saarma, Nature, 2007
Use of growth factors Therapeutic proteins (EPO, insulin, GDNF etc.) Research (especially developmental biology, neurobiology, stem cell research etc.) Antibodies to growth factors and their receptors are currently used for the treatment of cancer (EGFR, VEGF) and pain (NGF) Growth factors and their receptor are markers for disease diagnostics (cancer, neurodegeneration etc.)
Growth factors Most growth factors are normal secretory proteins Several growth factors are secreted as progfs and are therefore activated outside the cells by extracellular protease cleavage ProGFs may have different functions compared to mature growth factors Some growth factors have no known secretion signal (CNTF) Some growth factors are membrane bound proteins (ephrins)
Growth factors have very different structures: GFLs are homodimers GDNF NRTN ARTN PSPN
Neurokines are monomers
Two domains of MANF and CDNF. Probably also monomers Parkash, Lindholm et al., 2009
Growth factor proteins Can be monomeric or dimeric proteins Can be homodimeric or heterodimeric proteins The dimers can be covalent or non-covalent Growth factors may need a third component for activation (heparan sulfate for FGFs, HGF/SF etc.) Some growth factors are active only when bound to ECM (HB-GAM)
Growth factor receptors Consisting of one or of several components At least one receptor component should be transmembrane Receptor activation is ligand triggered, but receptors are usually active also without the ligands (dependence receptors) Direct and indirect activation of the receptor
Growth factor receptors One ligand can bind to several receptors (NGF, GDNF) with similar or different activity (NGF) One receptor can serve as the receptor for several ligands (p75, RET) Some receptors may have different functions in the complex with other receptors (p75/trk for mature neurotrophins and p75/sortilin for proneurotrophins) Ligated and unligated receptors may induce different signalling and cellular outcome (lifedeath for Ptc or RET)
Many mechanisms for ligand receptor interaction In most cases monomeric or dimeric ligand triggers signalling receptor dimerization and activation Ligand often bind to co-receptors and then the ligand co-receptor complex binds to a transmembrane signalling receptor Sometimes many ligands bind to a single receptor Sometimes ligand signals to the receptor and vice versa (bi-directional signalling for Ephrins and their receptors)
Growth factors signal into the cells via transmembrane receptors Signalling
Signalling
Signalling
Many HB-GAM or GDNF molecules bind simultaneoulsy to Syndecan-3 and activate it
Signalling by TNFRs Activated TNFR binds adapter molecules: TRAF1-6 and/or DD molecules caspase activation (apoptosis) NF- B (cell survival) JNK
Signaling pathways activated by CNTF neurotrophic factor Reichardt and Farinas 1997
General features of Eph receptors and ephrins
Growth factors active multiple signalling pathways that are integrated in the cells Neurotrophin receptor signalling
Growth factors regulate almost all aspects of cellular life Cell proliferation Cell migration Cell motility Cell differentiation Cell survival Growth factors have stimulatory and inhibitory effects (inhibit axonal growth, cell migration etc) Growth factors control almost every aspect of organisms development, maintenance and functioning
Discovery of growth factors
The Nobel Prize in Physiology or Medicine 1986 "for their discoveries of growth factors" Stanley Cohen Rita Levi-Montalcini
Viktor Hamburger 1900-2001
How growth factors were discovered? Early (1934) experiments by Victor Hamburger with chick embryo limb innervation showed that limb contains a substance that stimulates nerve growth Similar results were obtained by Rita Levi-Montalcini in the University of Turin in Italy during the war Elmer Bueker grafted fragments of mouse sarcoma 180 in to the body wall of three-day chick embryos. The histological study of the embryos fixed 3-5 days later, showed that sensory nerve fibers emerging from adjacent dorsal root ganglia had gained access into the neoplastic tissue while no motor nerve fibers entered into the tumor Victor and Rita then found that sympathetic and sensory ganglia were larger: tumor tissue stimulates nerve growth
A gift from malignant tissue: transplanted mouse sarcome 180 induces nerve growth and increases the volume of ganglia
Tumor tissue induces massive nerve growth and enlargement of ganglia: ganglia have more neurons
International collaboration Rita Levi-Montalcini. Attempts to replicate these effects by implanting dried tumor pellets or by injecting extract of either sarcoma were unsuccessful. Lack of facilities in this field in the Department of Zoology at Washington University, prompted me to ask hospitality from Professor Carlos Chagas, Director of the Biophysics Institute of the University of Brasil in Rio de Janeiro. There, a friend of mine, Hertha Meyer, had built and was director of a most efficient tissue culture unit. Upon approval and invitation by Professor Chagas, I boarded a plane for Rio de Janeiro, carrying in my handbag two mice bearing transplants of mouse sarcomas 180 and 37
Carnival in Rio: mouse sarcoma 180 induces neurite outgrowth from sensory ganglia direct contact of tumor and ganlia is not needed! A control B 24 hours C 48 hours What is the chemical nature of the factor?
What is stimulating nerve growth? Discovery of the Nerve Growth Factor, NGF Rita Levi-Montalcini in USA in 1946 Chick embryo sensory ganglia stimulated by NGF
Snake venom is a rich source of the factor A young biochemist, Stanley Cohen in St. Louis isolated from the two tumors a nucleoprotein fraction endowed with the in vitro nerve growth promoting activity. In order to degrade the nucleic acids present in this active fraction, Stan made use of snake venom which contains, among other enzymes, also the nucleic acid degrading enzyme, phosphodiesterase. Since a dense fibrillar halo was produced also around ganglia cultured in the presence of minute amounts of snake venom alone, it became apparent that the venom itself was a most potent source of nerve growth promoting activity.
An active protein was purified Stanley Cohen was in fact able to show that equivalent growth stimulation effects were obtained by 15,000 µg of a sarcoma 180 homogenate and 6 µg of the moccasin snake venom Nerve growth promoting activity, identified as a protein molecule with a molecular weight in the order of 20,000
Mouse submandibular salivary glands are rich in two factors: NGF and EGF Mouse submandibular salivary glands were found to be a very rich source of the nerve growth factor (NGF) S. Cohen then purified mouse NGF and determined its chemical structure, including the protein sequence Its availability in larger quantities than the venom NGF, and its moderate toxicity when injected in a highly purified form, made possible the exploration of its biological activity in neonatal, young and adult mammals Another growth factor, epidermal growth factor (EGF) was also purified from the same tissue by Dr. Cohen
NGF structure and interaction with receptors
The Neurotrophic Factor Hypothesis developed by Levi-Montalcini and Hamburger Holds true for the peripheral nervous sytem
Amino acid sequence of EGF was determined in 1972 the second growth factor after NGF
Identification of EGR receptors and internalization
Stimulation of EGF of the incorporation of 32 P-phosphate in A-431 cell membrane first evidence for EGF (growth factor) signalling
General principles of growth factor action
Growth factors are secretory proteins Growth factors are secretory proteins that bind to the specific receptors on the cell surface and activate them Some of the growth factors are membrane bound and are activated upon cleavage Some growth factors require cofactor for activity and some are active only, when bound to ECM Typically growth factors are secreted by well established pathways, but for several growth factors it is not known how they get out of the cells (CNTF, some FGFs etc.) Some growth factors are secreted constitutively and some in a regulated fashion One growth factor can be secreted in both ways
The structure of the rat BDNF gene and protein
Biological meaning of the regulated secretion Allows secretion of proteins in large amounts in a short time period (synaptic regulation) Allows to achieve high concentrations of the growth factors locally in synaptic cleft Allows also targeted secretion of the growth factors in cellular compartments (spines, growth cones etc.) Allows regulation of the secretion Reminds the mechanism of action of neurotransmitters
The route of BDNF from synthesis to secretion
Structural differences and similarities between the growth factors: prediction of the biological action and receptor is still very difficult
Growth factor receptors Receptor tyrosine kinases Receptor serine-threonine kinases Many others, where the intracellular domain has no intrinsic catalytic activity Receptors consist of one protein component, two or even three components Receptor activation is triggered by ligand (growth factor binding) Uncoupled receptors are also active
Neurotrophic factors and their receptors A NGF NT-3 BDNF NT-4 p75 TrkA TrkC TrkB B C GDNF NTN CNTF LIF CNTFR GFR GFR gp130 LIFR gp130 LIFR Ret
Models of Trk and p75 receptor activation From M. V. Chao
The transforming growth factor (TGF- )/SMAD pathway Binding of a TGF- family member to its type II receptor (1) in concert with a type I receptor (2) leads to formation of a receptor complex (3) and phosphorylation of the type I receptor (4). Thus activated, the type I receptor subsequently phosphorylates a receptor-regulated SMAD (R- Smad) (5), allowing this protein to associate with Smad4 (6) and move into the nucleus (7). In the nucleus, the SMAD complex associates with a DNA-binding partner, such as Fast-1 (8), and this complex binds to specific enhancers in targets genes (9), activating transcription.
Activation of receptor tyrosine kinases
GDNF interactions with the receptor
Proteins binding to receptor tyrosine kinases
From RTKs to transcriptional regulation
The NGF family and its receptors Purves, 22:15
Growth factors signal into the cells via transmembrane receptors The Shh signaling pathway involves two transmembrane proteins, Patched (Ptc) and Smoothened (Smo). Shh binds to Ptc, whereas Smo acts as a signal transducer. In the absence of ligand, Ptc interacts with and inhibits Smo. This inhibition activates a transcriptional repressor (e.g. Gli in vertebrates). In the presence of ligand, the interaction of Ptc and Smo is altered and Smo is no longer inhibited. Gli protein may then enter the nucleus and function as a transcriptional activator.
Growth factor receptors and their signalling complexes
What are lipid rafts? heterogeneities in biomembranes highly dynamic lipid-protein assemblies: composition, size and life-time variable lateral organizations held together by weak interactions based on 1) lipid - lipid immiscibility (cholesterolsphingolipids) 2) protein - lipid interactions
Lipid rafts Lipid rafts are plasma membrane microdomains rich in cholesterol and sphingolipids, which provide a particularly ordered lipid environment. Rafts are enriched inglycosylphosphatidylinositol (GPI)-anchored proteins, as well as proteins involved in signal transduction and intracellular trafficking
Rafts are implicated in many cellular functions: membrane trafficking cell polarity signal transduction cell adhesion cell migration pathogen invasion
Potential mechanisms for raft clustering
EGFR is moving out the raft
Lipid rafts and neurons In neurons, lipid rafts act as platforms for the signal transduction initiated by several classes of neurotrophic factors, including neurotrophins and glial-derived neurotrophic factor (GDNF)-family ligands Emerging evidence also indicates that such rafts are important for neuronal cell adhesion, axon guidance and synaptic transmission Lipid rafts are structurally unique components of plasma membranes, crucial for neural development and function
Trk receptors are activated in lipid rafts
GFR receptors are GPI-anchored proteins and after lipase cleavage they are found also in soluble forms
GDNF family ligands recruit and activate RET in rafts
GDNF recruits RET into lipid rafts via soluble GFR 1 and activates Src and FRS2 pathways
Ligand-induced endocytosis of the receptors: controls the balance and targets the intracellular signalling
Retrograde and anterograde axonal transport of BDNF
The route of BDNF from synthesis to secretion Neural activity triggers BDNF secretion. To keep a neuron alive neuron must signal!
Targeting of BDNF to dendrites
During development neurons form networks that are maintained and remodelled Neuron 1 Neuron 2 Neuron 1 fires to Neuron 2 Neuron 2 fires to Neuron 3 Neuron 4 fires to Neuron 1 Neuron 3 fires to Neuron 4
Neuron 1 fires to neuron 5 and triggeres the secretion of neurotrophic factors that maintain their connection Neuron 1 Neuron 1 secretes GDNFor BDNF in an activity-dependent fashion New connection with neuron 5 Neuron 4