MCDB 436 (11): Intracellular Signaling (1)

three stages of signaling

- reception

- transduction - needs propagation and amplification

- response

different parts of a cell involved in signaling

- membrane receptors: transfer information
- 2nd messengers: relay info from receptor ligand complex
- phosphorylation: common means of information transfer

phosphorylation

the major mechanism for intracellular signal transduction
- involves transfer of gamma-phosphate groups from ATP to AA sidechains of serine, threonine, and tyrosine in proteins

does not necessarily mean activation; not permanent

kinases

mediate the transfer of phosphate onto the protein

phosphatases

enzymes that remove a phosphate from their targets to flip proteins back into their non-phosphorylated state

characteristics of kinases and phosphatases

- some: soluble cytoplasmic proteins
- some: integral membrane proteins
- activity frequently regulated by the phosphorylation, leading to cascades

Ras/MAP kinase signaling transduction pathway

best characterized signaling cascade turned on by activated protein-tyrosine kinases (PTK)

activated receptor -> activate Raf -> phosphorlates MEK -> phosphorylates ERKs -> phosphorylate and activate target molecules (transcription factors, etc)

MAPK (miitogen-activated protein kinase) cascade

Raf (MAPKKK), MEK (MAPKK), and ERK (MAPK) signaling pathway

proto-oncogenes

overactive forms are associated with cancer
- for Raf, growth factor receptor + c-Myc

cell surface receptors

embedded in plasma membrane or embedded within intracellular space on a surface
1) GPCRs (G-protein coupled receptors)
2) RTKs (receptor tyrosine kinases, or enzyme coupled receptor)
3) ion channels

intracellular receptors

receptors in cytosol or nucleus
1) steroid or thyroid hormone receptors
2) some TLRs

GPCRs (G-protein coupled receptor)

contain seven (7) tansmembrane domains/helices; largest superfamily of proteins encoded by animal genomes

- amino terminus: outside of the cell. carboxyl-terminus: inside of the cell
- 7 alpha helices connected by loops, which are also binding domains
- binds hormones, NTs, opium derivatives, chemoattractants, odorants, tastants, photons

G-protein GPCR interaction

ligand binding -> GPCR activation -> turns on G proteins
- G protein acts as an on/off switch
- GTP bound = active; GDP bound = inactive

(photo: B2-AR [GPCR] + heterotrimeric G protein)

G proteins

heterotrimeric consisting of alpha, beta, and gamma subunits
- held at the plasma membrane by lipid chains covalently attached to the alpha and gamma subunits
- bind GDP (inactive) and GTP (active)
- consist of Gs, Gq, Gi, and G12/13

Ga (G protein)

the subunit for the guanine nucleotide-binding site

GTP -> Ga conformational change -> low affinity for Gbeta/gamma -> Ga dissociates -> free to activate an effector protein (such as adenylyl cyclase) -> effect, such as 2nd messenger

Gs - family G proteins

couple receptors to adenylyl cyclase

Gq - family G proteins

contain Ga subunits that activate PLCbeta

PLCbeta

hydrolyzes phosphatidylinositol bisphosphate -> produces inositol triphosphate (IP3) and diacylglycerol (DAG)

Gi - family G proteins

inhibit adenylyl cyclase
(i for inhibit)

G12/13 - family G proteins

inappropriate activation associated with excessive cell proliferation and malignant transformation

GPCR signal cascade steps

- rest: G protein in GDP state associates with membrane through Ga and Gy covalent binding to lipids

- step 1: ligand binding

- step 2: conformational change in GPCR -> binding pocket for Ga

- step 3: GTP-bound Ga dissociates from R*, releasing Ga-GTP and Gbeta/gamma to trigger effector proteins

- step 4: intrinsic GTPase activity of Ga converts Ga-GTP->GDP

- step 5: reassembly of heterotrimeric G protein

GPCR disorders

- constitutive stimulation (gain of function): 1, 2, 5, 6, 7, 8 - majority
- block (loss of function): 3 & 4

receptor tyrosine kinases (RTKs)

receptors activated directly by extracellular growth and differentiation factors (EGF, PDGC) or metabolic regulators (insulin)
- involves ligand-mediated and receptor-mediated dimerization
- dimerization results in joining of the two tyrosine kinase dmains -> trans-autophosphorylation (one activates the other and vice versa)

EGF and PDGF

epidermal growth factor :: platelet-derived growth factor

act as ligands on RTKs

ligand-mediated dimerization

dimer joins the two tyrosine kinase proteins

receptor-mediated dimerization

two ligands bind to two receptors, which then join each other

scaffold proteins

large proteins that can recruit other proteins (ex: becomes tyrosine phosphorylated on multiple sites to recruit)

adaptor proteins

membrane-anchored or cytoplasmic proteins containing several signaling modules that serve to link two proteins together

pTyr-binding domains

SH2 and PTB domains
- allow for association with activated RTKs since they can bind specifically to phosphorylated tyrosine residues

proteins that use SH2 or PTB domains

- adaptor proteins (Grb2)
- docking proteins (IRS)
- transcription factors (STAT)
- signaling enzymes (PLC-gamma)

forms of recruitment for signaling proteins to the membrane

- tyrosine phosphorylation of membrane associated proteins -> recruit SH2/PTB domain proteins, which also protects scaffold from dephos

- lipid modifications (Ras associating with the membrane, later activating)

- modifications to the membrane itself resulting from receptor activation (PIP3 [active form of PIP2] recognized by PH domains of protein kinases)

types of ion channels

- voltage-gated - Na+, K+, Ca2+
- ligand-gated - nicotinic AChR, AMDA/NMDA/glutamate, GABAa, glycine
- misc: mechanisensitive, thermosensitive

TRPV1

capsaicin receptor, a type of thermosensitive channel for noxious heat
- encoded by TRPV1 gene

second messengers

small, non-protein molecules that pass along a signal initiated by the binding of a ligand to its receptor
- transmit information by a change in their concentration; amplify the strength of the signal
- can be cyclic nucleotides (cAMP), metal ions (Ca2+), or inositol phosphates (phospholipids)

4 classes of second messengers

- cyclic nucleotide (cAMP)
- membrane lipid derivatives (IP3, DAG)
- Ca2+
- NO or CO

cAMP

2nd messenger synthesized from ATP by adenylyl cyclase (AC)
- binding of hormone to its receptor activates G protein -> activates AC

leads to response by either:
1) protein kinase A (PKA)
2) binds to cAMP response element binding protein (CREB)

PKA (protein kinase A)

a cAMP-dependent protein kinase that phosphorylates target proteins

CREB (cAMP response element binding protein)

a protein that binds to cAMP to control transcription of genes

mechanism of receptor-mediated activation and inhibition of cAMP

1) ligand binds to receptor -> conformational change -> increased G protein affinity
2) Ga gets GDP->GTP
3) Ga dissociates from Gbeta/gamma -> binds to effector (adenylyl cyclase) -> activate
4) adenylyl cyclase produces cAMP

5) GTPase gets GaGTP->GDP
6) Ga reassociates with Gbeta/gamma -> reforms trimeric G protein -> effector stops
7) cytoplasmic domain of the activated GPCR is phosphorylates by GRK
8) phosphorylated receptor has been bound by an arrestin molecule -> inhibits ligand bound receptor from activating additional G proteins

GRK (G-protein coupled receptor kinase)

a kinase that phosphorylates the cytoplasmic domain of activated GPCR
- involved in the receptor-mediated activation and inhibition of cAMP

desensitization

an arrestin molecule binds to a phosphorylated receptor, inhibiting the ligand bound receptor from activating additional G proteins

arrestin-mediated internalization of GPCRs

1) arrestin-bound GPCRs (aka desensitized GPCRs) internalized in clathrin-coated pits -> bud into cytoplasm
2) buds transformed into vesicles -> contents to endosomes
3) arrestins serve as scaffolds for assembly of signaling complexes
leads to...
4) GPCRs delivered to lysosome for degradation
OR...
4) GPCRs returned to the plasma membrane in a recycling endosome
5) interact with new extracellular ligands

glucose mobilization

example of response induced by cAMP
binding of hormone -> activation of enzyme and formation of cAMP -> cAMPbinds to PKA -> activates -> PKA phosphorylates:

1) phosphorylase kinase - phosphorylates glycogen phosphorylates, stimulates glycogen breakdown
2) glycogen synthase - inhibition prevents conversion of glucose to glycogen

Ca2+

2nd messenger; stored in ER (transported by Ca2+ pump)
- concentration is low in cytosol but higher in extracellular space or lumen of ER. kept low because:

1) Ca2+ channels in plasma membrane and ER membranes are kept closed
2) energy-driven Ca2+ transport systems of the plasma and ER membranes pump Ca2+ out of the cytosol

fluorescent indicator of Ca2+

1) chemical Ca2+ indicator (by laser)
2) bioluminescent protein
3) genetic encoded calcium indicator
4) FRET-based

IP3/DAG - Ca2+ signaling

PI -> (kinase) PI(4)P -> (kinase) PI(4,5)P2 -> G protein activation -> PI(4,5)P2 to PI-PLCbeta -> release IP3 -> IP3r -> ca2+ release from ER -> cytosol Ca2+ increase

PI-PLCbeta also creates DAG to recruit protein kinase PKC to the membrane

calcium-induced calcium release (muscle activity)

depolarization -> voltage-gated Ca2+ channels -> Ca2+ into cytosol -> bind to RyRs in SER membrane -> Ca2+ into cytosol -> cell contraction -> removed by SERCA and Na/Ca2+ secondary transport system -> relaxation

nitric oxide (NO)

can be an extracellular ligand or intracellular 2nd messenger

1) ACh binds to AChR
2) ligand-receptor -> increase Ca2+ concentration
3) Ca2+ activates NO synthase (NOS)
4) NO in endothelial cell diffuses across plasma membrane into adjacent smooth muscle cells
5) NO -> guanylyl cyclase (which makes cGMP) -> cGMP binds to cGMP-dependent PKG -> phosphorylates substrates -> relax muscle cell and dilate blood vessel

fine tuning response of the cascade response

- amplification of signal
- contribute to specificity of response
- provide opportunities to regulate signaling cascade

ex: rhodopsin absorbs photon -> 500 G-protein molecules activated -> 500cGMP activated -> 10^5 cGMP molecules hydrolyzed -> 250 Na+ channels close -> Na+ ions prevented (106/7) -> membrane potential altered by 1mV

rhodopsin and opsin

opsin linked to 11-cis-retinal; light induces isomerization of 11-cis to all-trans-retinal -> activates transducin (Gt)

cell signaling specificity

- different cell types have different set of proteins
- different protein profile gives each cell type specificity in detecting and responding to signals
- response of a cell to a signal depends on a cell's protein set
- pathway branching and cross-talk further help cells coordinate and regulate response to incoming signals

extracellular messengers

- AAs and AA derivatives (glu, gly, ACh, dop, etc)
- gases
- steroids (cholesterol derivative)
- eicosanoids (nonpolar molecules from FA named arachidonic acid)
- polypeptides and proteins

extracellular messenger receptors

- GPCRs
- RTKs (translate extracellular messenger molecules into changes in the cell)
- ligand-gated channels
- steroid hormones (ligand-reg'd transcription factors)
- unique receptors (BcRs, TcRs)