Describe the types of intracellular communication.
- contact-dependent: via membrane-bound signal mol.
- paracrine: mol. released by one type of cell, act on another type
- synaptic: neurotransmitters released to transmit electrical signals
- endocrine: hormones released into blood, affect distant cells
- autocrine: mol. released act on same cell/cells of same type
- gap junctions: intracell. mol. less than 1200 Da, electrical signals
Which 4 features characterize a receptor?
- high affinity to a special ligand (or substrate analogue) ∽ 10-9 mM
- can be saturated
- reversible binding
- evoke a biological response
What is Kd?
Formula.
defines concentration range in which receptor works best
→ concentration at which 50% of [L] bound to receptor
Kd = [R]*[L] / [RL]
- [R] = receptor concentration
- [L] = ligand concentration
- [RL] = receptor-ligand complex concentration
concept similar to Km <em>(cf. biochem)</em>
What is EC 50?
What is the difference to Kd?
efficient concentration that evokes 50% of the biological response
CAN be = Kd, but doesn’t have to (cf. potency, efficacy)
What is the difference btw potency and efficacy?
-
potency: amount of ligand required to produce an effect of given intensity
→ the ↓ [L], the ↑ potency -
efficacy: ability of ligand-receptor complex to produce a maximum biological response
→ the ↑ response, the ↑ efficacy
What is the difference btw agonist and antagonist?
- agonist → biological response
- antagonist → NO biological response
<u>DO NOT confuse pharmacological/muscular antagonists: </u><br></br><strong>- pharmacol. ant.</strong> inhibit agonist <strong>w/o causing an own effect</strong><br></br>- muscular ant. cause opposite movement
What is basal activity?
activity of receptor even in the absence of a ligand
Explain different types of agonists and antagonists.
dependent on efficacy
- full, partial agonists: evokes biological response
- antagonist (= neutral)
- inverse agonist: inhibits biological response to a level below basal activity
Briefly explain the function of a pharmacological agonist in case of diabetes insipidus.
diabetes insipidus = lack of ADH (= vasopressin)
→ no reabsorption
⇒ very high V, very low Osm urine excreted (- 20l/d)
⇒ vasopressin-agonist desmopressin administered in form of nasal drops
(would be degraded in intestines if administered per os)
Why is desmopressin instead of vasopressin administered in case of diabetes insipidus?
- vasopressin binds to V1 and V2 receptor
- desmopressin only binds to V2 → no add. vasoconstriction that would incr. blood pressure
What are 2 general classes of receptors?
What are their substrates?
- plasma membrane receptors: ligands that cannot cross plasma membrane
- nuclear receptors: small hydrophobic mol. (steroid hormones, thyroid hormones, retinoids, vit D)
What are 2 types of signal transduction pathways of plasma membrane receptors?
intracell. signals = molecular switches
active - inactive form
- signaling by phosphorylation: phosphorylation = activation (ATP → ADP + P, P transferred to substrate)
- signaling by GTP-binding protein: binding of GTP to effector = activation
reverse effects (dephosphorylation, hydrolysis of GTP to GDP) cause inactivation
What is adaptation?
Other terms.
also: desensitization
reversible reduction (= internalization)/inactivation of receptors due to prolonged exposure to ligand, associated w/ β-arrestin
What are types of plasma membrane receptors?
- G protein-coupled receptors (GPCRs)
- ion channel receptors
- receptors w/ enzyme activity
- enzyme activity-linked receptors
Explain the structure of G proteins.
- 7 transmembrane domains
- heterotrimeric: α, β, γ-complex in resting state
- in resting state GDP bound to α-subunit
Explain the function of G proteins.
What are GEFs and GAPs?
- GEFs: facilitate exchange of GDP to GTP at α-subunit of G protein (activate G proteins)
- GAPs: enhance hydrolysis of GTP to GDP + P (inactivate G proteins)
List some important G protein families.
- Gi/0
- Gq/11
- G12/13
- Gs
- Gt
List effects of the Gi/0 protein family.
- inhibition of adenyl cyclase
- activation of K+ channels → hyperpolarization
- inactivation of Ca2+ channels → no Ca2+ influx
- phospholipase A2 activation
Explain the cyclic AMP system.
Which family of G proteins stimulate, resp. inhibit it?
- a) GS stimulate
b) Gi/0 inhibit adenyl cyclase - adenyl cyclase: ATP → cAMP (= second messenger)
- activates PKA
- a) phosphorylates proteins → direct effect
b) enters nucleus, activates transcription factor CREB → indirect effect
Which enzyme degrades cAMP?
To … ?
Example for an inhibitor.
cAMP diphosphoesterase:
cAMP → 5’ AMP
caffein = inhibitor
Which receptors stimulate GS proteins?
β-adrenergic receptors, ACTH receptors (adrenocorticoreceptors)
Which receptors stimulate Gi/0 proteins?
α2-adrenergic receptors, M2, M4 ACh receptors, opiate (μ, δ, κ) receptors
Where do phospholipase A1, A2, C, and D cleave phospholipids?
- phospholipase A1: cuts ester bond at C1
- phospholipase A2: cuts ester bond at C2
- phospholipase C: cuts just before phosphate group
- phospholipase D: cuts just after phosphate group
Which G proteins activate phospholipase A2.
Explain its function.
activated by Gi/0
releases arachidonic acid from plasma membrane to be metabolised
List 3 metabolic pathways of arachidonic acid and their products + function.
COX produce
- thromboxanes: platet aggregation, vasoconstriction
- prostaglandins: platelet aggregation, bronchoconstriction, induce inflammation
- prostacyclins: inhibit platelet aggregation, vasodilation
5-lipoxygenase produces
- leukotrienes: allergic/inflammatory response (e.g. asthma, rheumatoid arthritis)
epoxygenase produces
- HETE, EET: incr. Ca2+ release from ER, stimulate cell proliferation
What are inhibitors of COX 2?
Function?
- *non-steroidal antiinflammatory drugs**,
e. g. aspirin, ibuprofen
Explain the function of Gt protein transducin.
activates phosphodiesterase in rod cells of the eye in response to light to convert cGMP to GMP
→ closes cGMP activated cation channels
⇒ altered membrane voltage
Which receptors stimulate Gq/11 proteins?
α1-adrenergic receptors, M1, 3, 5 ACh receptors, angiotensin II receptors
Which G proteins activate phospholipase Cβ.
Explain its function.
activated by Gq/11
converts PIP2 → InsP3 (IP3) + DAG
- InsP3 → binds to ER → opens Ca2+ channels
- DAG → activates PKC → phosphorylates proteins
What are ways for Ca2+ to enter the cell other than binding of InsP3 to the ER membrane?
- via voltage sensitive-Ca2+ channels
- via other ligand gated-Ca2+ channels
What are store-operated Ca2+ channels?
Relate the concentration of Ca2+ in ECF, cytoplasm and ER.
located in ER membrane, open if Ca2+ content in ER too low
(in ECF and ER 10-3M, cytoplasm 10-7M)
Explain the role of Ca2+ as second messenger.
Give examples.
- binds to calmodulin CaM
- activates CaM kinases, e.g.
- cAMP phosphodiesterase (cf. cAMP degr.)
- myosin light-chain kinase → sm. mm. contraction
Which receptors stimulate G12/13 proteins?
What is their function?
thrombin receptors, angiontensin II receptors
⇒ activate Rho
Classify small G proteins.
Function?
also regulated by GAPs, GEFs
Classify adrenergic receptors according to:
- subtypes
- affinity
- G protein
- signaling pathway
and name one example.
NE = norepinephrine
E = epinephrine
Explain the function of ligand gated-ion channel.
Example.
signal molecule binds to channel → opens
e.g. acetylcholine binds to nicotinic ACh receptors at neuromuscular junction → Na+/K+ channels open
What do all excitatory, and all inhibitory ligand gated-ion channels have in common?
List examples.
- excitatory: ion channels specific to cations
- inhibitory: ion channels specific to anions
What are the 2 general types of acetylcholine receptors?
- nicotinic acetylcholine receptors (nAChRs) = ligand gated-ion channels
- muscarinic acetylcholine receptors (mAChRs) = G protein-coupled receptors
What types of nicotinic acetylcholine receptors nAChRs are there?
List their specific inhibitors.
- muscle type: mediates skeletal mm. movement, inhibited by curare
- neural type: in symp./parasymp. ganglia, inhibited by ganglial blockers
What types of muscarinic acetylcholine receptors mAChRs are there?
List their common inhibitor.
To which G proteins are they coupled?
What is their action?
5 subtypes, M1 - M5 - all inhibited by atropine
- M1, 3, 5 coupled to Gq/11 → Ca2+, InsP3, PKC
- M2, 4 coupled to Gi/0 → cAMP ↓, K+ ↑, Ca2+ ↓
What are the 3 types of receptors w/ enzyme actitivity?
Examples for each.
- receptor guanylyl cyclases: ANP receptor
- receptor threonine/serine kinases: TGF-β receptor
- receptor tyrosine kinases: NGF receptor, insulin receptor
What are the types of receptor guanylyl cyclases?
What is their common function?
- membrane bound receptor guanylyl cyclases
- soluble receptor guanylyl cylclases
⇒ convert GTP to cGMP
Explain the function of the receptor that binds ANP.
What type of catalytic receptor is it?
GC-A = membrane bound-receptor guanylyl cyclase
- structure: ligand receptor, kinase-like domain that binds ATP, catalytic domain
- ligand = e.g atrial natriuaretic peptide ANP
- converts GTP to cGMP
→ here: inhibited Na+/H20 reabsorption by collecting duct
Explain the function of soluble receptor guanylyl cyclases referring to an example.
- structure: only catalytic domain w/ α- and β-subunit
- activated by intracellular NO
- converts GTP to cGMP
→ here: intracellular ↑Ca2+ binds to CaM → activates NO synthase to produce NO from Arg → activates soluble GC → cGMP relaxes smooth mm.
nitrogylcerin increases NO production → former treatment for angina pectoris
Explain the function of the TGF-β receptor.
What kind of catalytic receptor is it?
threonine/serine kinase
- structure: type I, type II subunits, multimeric
- type I activates downstream effectors
TGF-β binds to type II → phosphorylates type I → activates R-Smad → forms dimer w/ Co-Smad → translocates into nucleus to act. transcription factors
Explain the general structure of receptor tyrosine kinases w/r/t their function.
Examples.
- extracellular domain: cysteine-rich, iG-like, fibronectin type-III-like
- transmembrane domain: α-helix
- cytosolic domain: tyrosine kinase, can have tyrosine insert regions (= interrupted) w/ SH2 binding domain
ALWAYS: dimeric → autophosphorylation
→ different classes: many GF receptors, insulin receptors, etc.
Name some proteins that can directly bind to the SH2 domain of activated receptor tyrosine kinases.
Function?
- PI 3 kinase → phosphorylates lipids, causes inhibition of apoptosis (cf. own card)
- GAPs → enhance hydrolysis of GTP, inactivate Ras
- PLCγ → same function as PLCβ: PIP2 → IP3 + DAG
- SOS → Ras-GEF, part of Ras-MAP kinase signaling pathway (cf. own card)
Explain the function of receptor tyrosine kinases in the Ras-MAP-kinase signaling pathway.
- Grb2 recognizes SH2 domain of phosphorylated tyrosine on the activated receptor
- recruits SOS by means of two SH3 domains
- SOS (= Ras-GEF) stimulates inactive Ras to replace its bound GDP by GTP
- activates Ras to relay the signal to MAP kinases (cf. own card)
Explain the MAP kinase cascade triggered by Ras.
What is the final consequence?
- Ras activates MAP kinase kinase kinase Raf
- Raf activates MAP kinase kinase Mek
- Mek activates MAP kinase Erk
- Erk enters nucleus to activate transcription factors
Explain the pathway of PI 3-kinase activated by receptor tyrosine kinases.
- extracellular survival signal activates RTK, recruits + activates PI 3-kinase
- PI 3-kinase produces PI(3,4,5)P3 (= docking site for 2 serine/threonine kinases) w/ PH domains
- PDK1 and Akt bind to PI(3,4,5)P3 on plasma membrane
- phosphorylation + activation of Akt by PDK1 and mTOR → dissociates from plasma membrane
- Bad is phosphorylated by Akt → releases apoptosis-inhibitory proteins
⇒ promote cell survival
(phosphorylated Bad binds to a ubiquitous cytosolic protein → keeps Bad out of action)
What are enzyme activity-linked receptors?
Examples.
receptors which don’t have intrinsic kinase activity, but associate enyzmes
⇒ are dimeric
e.g. receptors for GH, prolaction, cytokines
Explain the function of the GH receptor.
- receptor dimerizes in response to GH binding
- binds 1+ JAK tyrosine kinases → phosphorylate themselves and the receptor
- STAT tyrosine kinases bind to complex + are phosphorylated
- STATs dissociate as dimers + translocated to the nucleus → phosphorylate key transcription factors
Which proteins can be considered oncogenes?
oncogenes = genes that potentially cause cancer if mutated
- GF receptors
- Ras
- PI 3-kinase
- Grb2
Explain the function of intracellular receptors.
hormone either:
- binds to cytoplasmic receptor that enters nucleus after binding (e.g. cortisol, aldosterone)
- binds to nuclear receptor that is already in connection w/ DNA (e.g. thyroid hormones, steroids, vit D3, retinoic acid)
⇒ regulate transcription in 2 steps, early/late response
What are the 2 ways of signal termination of G proteins?
ligand disassociates, then
- α-subunit hydrolyses GTP, reassociates w/ βγ-subunit - facilitated by RGS proteins
- phosphorylation of C terminal by receptor (GPCR) kinase and subsequent binding of β-arrestin
→promotesreceptor internalisation
→ G protein cannot access receptor anymore = desensitization
Explain the process of receptor internalisation.
- arrestin connects receptor to internalisation protein clathrin
- coat assembly of clathrin around receptor
- bud formation
- vesicle formation, dynamin cleaves connection of receptor to membrane
- uncoating, vesicle loses clathrin coat
Explain the action of insulin upon GLUT 4 receptors.
insulin binding to insulin receptor causes to relocalization of GLUT 4 receptors to plasma membrane to boost glucose uptake