Regulated Secretion PDF

Regulated Secretion Classical Neurotransmitters: synaptic vesicles, Neuropeptides: Dense Core Vesicles (DCVs) What we will learn today • How are neuropeptides processed • Aggregation and timing of cleavage key for sorting neuropeptides into DCVs • Transporters key for sorting classical transmitter...

Regulated Secretion Classical Neurotransmitters: synaptic vesicles, Neuropeptides: Dense Core Vesicles (DCVs) What we will learn today • How are neuropeptides processed • Aggregation and timing of cleavage key for sorting neuropeptides into DCVs • Transporters key for sorting classical transmitters into synaptic vesicles ! !"#$%&'( )*+'(,*+ ($# "* .*/0.1*2 $#2 .*#,,*2 $& &$* +"#$%+*% 678+ CANNOT! Regulated secretion ! '$#$ %&'()$ )% *#'(+,# -) .,/'0/ 0#0"2/$# 2#(&(2#' / '.#+(#+ '-(0&,&' 5)..)'#6 -) +)$'-(-&-(*# '#+2#-()$7 ! )&,-(.,# -9.#' )% 2#:&,/-#6 '#+2#-()$ /2# ;$)<$ " !9$/.-(+ *#'(+,# 2#,#/'# " *#:&,/-#6 2#,#/'# )% *#'(+,#' 6#2(*#6 %2)0 #$6)')0#' 5#&:& @)+@ 2#+#.-)2 ($'#2-()$% ,,&+)'# -2/$'.)2-#27 " *#:&,/-#6 '#+2#-)29 *#'(+,#' 5D#$'# +)2#6 *#'(+,#'-!#+2#-)29 :2/$&,#'7& " .9')')0# )2 ,9')')0#/2#,/-#6 )2:/$#,,# 5#&:& )#,/$).$)2#7 secretion. Organization at the Synapse Presynaptic Terminal Synamptic vesicles are NOT transported down the axon, only pre-synaptic vesciles are. Synaptic vesicles are made and used LOCALLY there are two different types of receptors for synaptic vesicles: neurotransmitter gated channel —> really important for fast synaptic transmission NEUROPEPTIDES do not use this channel g-protein-gated receptors —> go through seceond messengers, and are slow channels -neuropeptides use g-gated receptors! Post-synaptic Spine Synaptic Vesicles (Neurotransmitters) Active Zone vesicles docked and released from active zone Post-synaptic Density (PSD) Neurotransmitte G-proteinGated Channel -gated Receptor core is the aggrgated Dense Core Vesicle dense crystal of the cekk (ie. insluin concentrated in beta (Neuropeptides) cells) Difference between ‘classical’ and peptide transmitters in neurons • Major differences are due to how they are made and how they get into vesicles. this is why they can be made locally at the synapse! • Classical transmitters are synthesized in the cytoplasm and then packaged into synaptic vesicles by transporters. Peptide transmitters are synthesized by translation and processed through ER and Golgi and packaged into regulated secretory vesicles at the TGN. Fast vs Slow Neurotransmission DOPINE ONLY MEDIATES SLOW TRANSMISSION, NEUROPEPTIDES ONLY MEDIATE SLOW TRANSMISSION. CLASSICAN TRANSMITTERS (IE. GLUTAMATE RECEPTORS) CAN DO BOTH!! • Classical transmitters are released at the active zone and may mediate fast or slow neurotransmission, while neuropeptides are released from outside the active zone and only mediate slow neurotransmission. • Fast Neurotransmission --> Ligandgated Ion Channels; start electrical signal. • Slow Neurotransmission --> G-protein linked receptors; start chemical signal by altering second messengers How are synaptic vesicles made • Synaptic vesicles do not bud de-novo from TGN, but instead synaptic vesicle proteins are transported with other axonal proteins in axonal transport vesicles. THEY ARE REUSED MANY TIMES • Synaptic vesicles form after endocytosis from plasma membrane or from endosomes through budding of clathrincoated vesicles. This is both de-novo can be filled with formation and recycling. They neurotrasmitters through – Can then be filled with transporters after endocytosis. What are regulated secretory vesicles/ dense core vesicles/secretory granules? • Endocrine cells and Neurons have a specialized pathway for regulated secretion. • In this pathway, proteins are sorted at the TGN into a special type of vesicle that has different names in different systems, but all refer to the same vesicle. • These vesicles are transported to release sites but are only released after a specific stimulus; either calcium entry in neurons or sometimes cyclic nucleotides in some endocrine cells. • Once peptides are release, cannot be recycled. Need new vesicle from TGN. Dense core vesicles can partly fuse ! !"#$ %#$&# '()# *#&+',#& -.&# /"#0 '1$ 21)/+1,,0 -.&# 3-.&+($ 2()#4 () you cannot store a neuropeptide inside a synaptic vesicles!! '(52,#/#,0 -.&# ! 6#$&# '()# 3$#.)(2#2/+%#&4 1)# $(/ )#,#1&#% 70 21)/+1, -.&+($8 7./ 7+(9#$+' 15+$#& '1$ 7# )#,#1&#% " :$ /"+& ;108 &(5# 6<=& '1$ 1'/ 21)/+1,,0 ,+># &0$12/+' *#&+',#& -() )#,#1&# (- &51,, /)1$&5+//#)&? " @(;#*#)8 /"#)# +& $( 1'/+*# A($#8 $( &0$12/+' ',#-/8 $( B-1&/C $#.)(/)1$&5+&&+($D this requires synaptic vescicles (think back on fusion lecture and how fast, “fast synaptic transmission” is). Lifetime of DCVs this is partial fusion (cavicapture) How are proteins sorted into regulated secretory vesicles. they aggregate into a dense core at TGN, and cells know how to sort aggregates. The aggregation is the likely sorting signal • Sorting by Entry – Aggregation (formation of the dense core); aggregates bind to receptor or lipid rafts. – Facilitated aggregation (binding to chromogranin or secretogranin) these will co-aggregate with the precursors this is the – Sorting receptor exception, not used for many • Carboxypeptidase E binding vesicles • Di-basic residue binding prohormone convertase – Association with lipid rafts • Sorting by Retention – Remove all other proteins from TGN as immature secretory granules are still a sorting compartment All Models probably play a role for some cargo sorting by retention = if you’re gonna go somewehre else, bud off secretory granule until everything that could bud off, did buf off, until you are left with aggregate Chromogranins • Chromogranins are acidic proteins that aggregate in the acidic environment of the if you aggregate too early, its hard to process thru ER and TGN. -golgi, so u aggregate in TGN • Removing Chromogranin A (CGA) using siRNA decreases number of DCVs; overexpression of CGA causes DCVs to be formed in cells lacking its not clear a regulated secretory system whethre CGA is – However, KO of CGA does not show in real animals, sufficient for peptide hormones regulating complete loss of secretory granuleshave some ability to secretion aggregate on their get SOME – Argument over whether DCVs in own secretion but nowhere near overexpressing cells are regulated secretory as good as a granules. regulatory secretory cell ! !,# $%&& '(%&& )**+,*)(, )-. /0+1 .,-', 20+,'3 "5( )+, ($,', +,)&&7 ',2+,(0+7 *+)-5&,'= Neuropeptide Precursor Structure neuropeptides are ALWAYS cleaved off a precursor protein SO you can have multiple neuropeptides on one prohorhome (proneuropeptide) -they will have di-basic (lyine/arginine) cleavage sites on either side of the neuropeptide Neuropeptide A Signal Sequence -they have signal sequence bc they have to be inserted into ER through secretory vesicle - Cleavage Sites 1-50 Peptides/Precursor thats only way to enter is to get co-translationally secreted into ER Neuropeptide B Glycine for Amidation some (but not all) have glycine signal that trigger an amide group to be added generally peptides that are travelling long distances will need amidation so they don’t get degraded. Local neurpeptides don’t need to last a long time, so they won’t have a glycine. Processing of Neuropeptdies Neuropeptide Signal sequence cleavage (ER) Neuropeptide Endoproteolytic cleavage (TGN or secretory vesicle Neuropeptide when cleavage happens, dibasic residues stay on and have to be removed bt carboxypeptidase Carboxypeptidase (secretory vesicle) Neuropeptide Peptidyl-glycine-a-amidation (vesicle) Neuropeptide NH (amide) 2 mature neuropeptide Different processing enzymes cleave in distinct compartments • Furin site (BXBB) B is basic amino acid is an enzyme that will cleave basic (Lysine or Arginine); Furin resides and NEEDS 3 basic residues to cleave – Furin cleaves at pH of TGN. • PC1 and PC2 site (BB) these need two basic residues to cleave – PC1 and PC2 cleave in immature secretory granules • Different peptides can be made in different neurons as some specificity in PC1 and PC2 sites. • Timing of cleavage is important in sorting of neuropeptides. Egg laying hormone is an interesting example of importance of timing ! !"#$%&"&'()" &$"*#$+%$ $-+ %." /#$(. +('"% *1"-2-3" (. '$" ,! +"&-$-'"+ &$"*#$+%$ (.'% '6% +()"+& *"+' %/ &"&'()"+ *1"-2") ": +<> -.) one side has hormone that goes into circulation, other side +<A (. (??-'#$" 3$-.#"1"+& has hormone that control firing of that neuron ! $" '6% +()"+ -$" +%$'") (.'% )(2"$".' A<B+ -' '$" ,!& " $" A<B+ )(2"$ (. '$"($ +(3"D 6$"$" '$": -$" '$-.+&%$'") '% -.) 6$"$" '$": /#+"& ! 7. *"11+ 6('$ 1"++ /#$(.D &$"*#$+%$ (+ .%' *1"-2") -.) &"&'()"+ -$" *%/+'%$") (. +-?" 2"+(*1"& Egg laying hormone precursor FURIN SITE signal seq. two sides aggregate seperately these sort into a different vesicle, secrete at Soma (cell body) this vesicle gets put into release sites, to get put into the blood Differential sorting of different peptides from same precursor to prove this, they had to create antibodies against all these hormones - and they had to make secondary antibodies in another species of animal it is clear some vesicles had SMALL gold and no Large gold. Other vesicles had only LARGE gold and not small gold in them Small gold: ELH Large gold: N-terminal peptides Aggregation is sufficient for DCV entry • The fact that different aggregates form separate granules, suggests that aggregation is sufficient for sorting. • This has been seen in other systems, and can often be seen by overexpression of a granule component. But what about transmembrane proteins that are sorted into DCVs? • Aggregation probably does not explain sorting of transmembrane proteins (like transporters) to DCVs. • Specific cytoplasmic signals have been identified in VMAT2, Phogrin, and the PMAT enzyme that converts glycines into N-terminal amides required for sorting into DCVs. ANSWER on next slide!! Sorting of transmembrane proteins into DCVs is NOT at the TGN! • These proteins are sorted in later from vesicles budding from endosome and fusing with immature dense core vesicles. – Many proteins involved in endosomal trafficking have been identified in genetic screens for neuropeptide secretion • Rab2 and its interactors • A specific Vamp-like protein (Vtia) • EARP complex (tethering proteins important for fusion) – 5"#$%& $# '&%(& )% "+ ,-$)$,./ 0%- 123 4.)"-.)$%& Sorting of transmembrane proteins into DCVs is NOT at the TGN! Neurotransmitters are packaged through transporters • Classical transmitters (Dopamine, Noradrenaline, Acetylcholine, Glutamate, GABA, glycine, serotonin, histamine) are synthesized by enzymes in the cytoplasm. • They are transported into synaptic vesicles through specific vesicular transporters. • Many share the same transporter – VMAT: Dopamine, Noradrenaline, serotonin, histamine – VGAT: Gaba, Glycine – Vacht: Acetylcholine – Vglut: Glutamate Vesicular Transporters and Evolution of nervous system • Regulated secretory vesicles predatepresent yeast +fungi! the evolution of synaptic vesicles. • Some of the synaptic vesicle transporters and ligand gated ion channels evolved before the nervous system • So, regulated secretion of ATP and glutamate probably started from DCVs and were ready for the ‘invention’ of synaptic vesicles. in Transporters are also in dense-core vesicles • There is no way for neuropeptides to get into synaptic vesicles that are formed by endocytosis at the terminal. • However, there is nothing to stop a transporter from being sorted into a DCV similar to other transmembrane proteins – Vesicular transporter for biogenic amines is in chromaffin granules – Vesicular transporter for ATP is in Vesicular and Plasma Membrane Transporters Vesicular Protein ATPase ATP you need energy (atp) to transport vesicles in - because you have to go against protein gradient. Use Vesicular ATPase, which acidifies vesicles and makes a pH gradient ADP H+ Enlarged Synaptic vesicle 5-HT Na + 5-HT selective transporter H+ Plasma Membrane Transporter non-selective transporter Vesicular Transporter -these are NOT drug targets, bc they are SYNAPTIC VESICLE transporters, not PM transporters What determines which transmitter a neuron uses. • For neuropeptides it is the expression of the peptide itself, perhaps with a specific Prohormone convertase. • For classical transmitters it is the specific synthetic enzymes to make the transmitter (i.e. choline acetyl transferase is only found in neurons that use acetylcholine as a transmitter; similar for glutamic acid decarboxylase for GABA), although no specific enzymes for glutamate and glycine which are present in all cells. all these have is vesicular tranporters! no enzyme • Specific vesicular transporter to uptake transmitter into vesicle (i.e. vesicular Dale’s Hypothesis • Neurons use only one transmitter WWRONG • While generally true and a useful concept, several caveats and modifications • True for classical transmitters only; neuropeptides are co-transmitters in many cells. • GABA and glycine have been shown to be released from some neurons from same vesicle (they share the same transporter). • Biogenic amines (dopamine, noradrenaline, serotonin, etc) all use the same transporter, but are differentiated by the synthetic enzymes present in each cell. Glutamate transporters argue against Dale’s hypothesis • The only protein needed to release glutamate as a transmitter is the transporter (abundant glutamate is present in all cells). • Glutamate transporters are not only present in glutaminergic neurons (most excitatory neurons), but many other neuronal cell types as well (dopaminergic, GABAergic, evidence suggests glutamate may be Serotonergic). Growing co-released at many synapses • Growing evidence suggests glutamate Re-use is a major difference between synaptic vesicles and dense-cored vesicles • Synaptic terminals are located at the end of the axon; far from the cell body of neurons. • Synaptic vesicles are formed from endocytosis after fusion, refilled with transmitter and thus are re-used many times. • Dense core vesicles must be reformed from the trans-golgi network in the cell translation of neuropeptides may allow local body. Local production of releasable DCVs? – Reuse of synaptic vesicles if you made DCVs locally, but you woudl have to LOCALLY translate them, and you need all the machinery for this. There is no evidence you can do this yet. Nature. 2014 Nov 13; 515(7526): 228–233. What we learned today • How are neuropeptides processed • Aggregation and timing of cleavage key for sorting neuropeptides into DCVs • Transporters key for sorting classical transmitters into synaptic vesicles • Synaptic vesicles can be reformed and refilled at the synapse; DCVs