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Types of cellular signals and receptors
- intracellular
- extracellular
Extracellular receptors
- receptors located on the surface of cells
- bind to extracellular ligands to activate intracellular cascades
Intracellular receptors
- receptors locate in the cytosol of cells
- lead to the activation of transcription
Overall, cellular signaling generally leads to
- protein synthesis
- initiation of transcription
How can the rates of cellular signaling responses vary from ptw to ptw?
- changes in proteins activity can be rapid
- changes in transcription are typically slower
- stability of synthesized proteins can determine rate of change (more stable: longer rxn/persistence of rxn)
The most common receptors are located on
the cell surface
Generic signaling ptw/outline
- ligand binds to extracellular/intracellular R
- R activates/leads to activation of intracellular proteins
- activated proteins lead to a signaling cascade
- effector proteins are activated, leading to changes at the cellular lvl (transcription, protein synthesis, ect.)
Intracellular Rs typically bind to what types of ligands?
- hydrophobic molecules
- pass through the CM to bind
- leads to initiation of transcription
Types of cell-cell communication
- contact dependent (direct)
- paracrine (local/short distances)
- synaptic (neurons and NTs)
- endocrine (use of blood stream to deliver ligand to target cell)
Endocrine signaling is involved in
coordinating overall organism physiology
Aspects of receptor functions
- specificity
- amplification
- desensitization/adaptation/feedback
- integration
- protein complex formation
- concentration dependent action
Specificity
- a receptor function/characteristic
- a specific signaling molecule fits a specific site on its complementary R
- other signals don't fit the binding site
Specificity of cellular receptors also extends to
- effector function/the end result of signaling (specificity is influence by both hormone and R signaling machinery)
- ex: ACh
- when ACh binds to the ACh R in different tissues, it elicits different responses (heart: decreased firing rate; muscle: contraction)
- Still the same ACh R
Amplification
- a receptor function/characteristic
- a single signal molecule can activate multiple proteins/enzymes
- signal transduction cascades
Example of amplification
- epinephrine (epi)
- when epi binds to its R, it activates X molecules,
- X # of molecules go to activate 20x of cAMP
- 20x of cAMP go on to activate 10x of PKA.......
Desensitization/adaptation
- a receptor function/activation
- essentially the process of turning off a signaling ptw
- involves a feedback loop from the activated R that leads to its own shutdown
Examples/methods of receptor desensitization/adaptation
- R sequestration
- R down-regulation/degradation
- R inactivation
- Inactivation of signaling proteins
- synthesis of inhibitory proteins
- different stimuli will induce different feedback loops (+ or -)
Integration
- a receptor function/characteristic
- the involvement of 2 ptws can have different effects on metabolic ptws
- idea of cross-talk: 2 dif signals can have the same/different effect on the same target
Integration is essentially the combination of
combination of signaling molecules that define the cellular response
Scaffold proteins
- protein complexes involved in cellular signaling
- directly organize signaling molecules to aid in amplifying a cellular signal
- sequester signaling molecules for efficiency in turning on the same ptw
Signaling complexes (scaffold proteins) can be assembled in what ways?
- they can already by associated w/the R
- can be recruited once the R becomes activated
- phosphoinositides can also serve as docking sites (typically by the CM)
- multiple docking strategies can be used @ once
How are concentrations/gradients involved in cellular signaling?
- [gradients] define responses to hormonal signaling
- induction of ptws can be dependent on [signaling molecules] (can be broad/narrow range of signal concentrations)
The classic dose response curve for signaling molecules and their concentrations is usually in the shape of
- a sigmoidal curve
- multiple signals are needed to shift the shape of the curve
- indicative of cooperativity in signaling
Types of receptors
- ligand gated ion channels
- nuclear receptors
- adhesion receptors
- receptors guanylyl cyclase (RGC)
- tyrosine kinase receptors (RTKs)
- G-protein couples receptors (GCPRs)
GCPRs
- Rs located in the PM
- binding of a ligan induces a conformational change
- activates G proteins which go off to activate signal cascades
RTKs
- ligand binds to R
- activated RTK activated tyrosine kinases via autophosphorylation
- leads to a kinase cascade (MAP cascasde)
- initiates upreg of TFs and enzymes
RGKs
- enzyme receptors
- bind ligands to activate
- activation leads to production of cGMP (GTP > cGMP)
RGKs can be desensitized by
- dephosphorylation
- cGMP breakdown (prevention of breakdown keeps response active)
Steroid (and gaseous) hormone Rs
- intracellular Rs that bind hydrophobic molecules
- Rs themselves act as TFs to regulate gene expression
- have unique DNA binding domains
- lead to primary and 2ndary transcriptional events
Since steroid/gaseous Rs are TFs themselves, ligand binding results in either
- ability of the R to bind to DNA
- ability of the R to recruit co-activating proteins that can bind to the R
The conformation of steroid/gaseous Rs is dependent on
- ligand binding
- coactivator protein binding
Primary and 2ndary transcriptional events of steroid/gaseous Rs
- primary: synthesis of primary proteins that go and shut off primary protein genes AND go upregulated 2ndary response protein genes
characteristics of ion channel signaling
- channel opening is gated and selective (ion filter)
- opening gates initiates signaling ptw
- ions move with their [gradient]
- ions must be at a [low cytoplasmic]
- channels are only transiently open
Ion gradients in cells are determined by
- primary active transport
- energy expensive movement of ions
Integrins
- a type of mechanically gated ion R
- bind the ECM to the CM
- integrated cellular activity by sending signals from the inside out and vice versa
Ion channels can be opened by what methods?
- ligand binding
- voltage gated (rely on membrane potential)
- mechanical (physical stress opening)
G protein coupled receptors (GCPRs/GPCRs)
- 7 passing tmem protein; serpentine
- utilizes g proteins that activate 2ndary molecules
GPCRs typically utilize what 2ndary messengers?
- cAMP
- DAG
- IP3
Generic function of GPCRs
- 1st messenger/ligand binds to the GCPR
- the GCPR activates > activates the G protein
- the G protein converts GDP > GTP
- G protein bound to GTP is a functional enzyme that can activate 2ndary messengers
- G protein-GTP activity leads to a cellular response
What defines the signaling outcome when GCPRs are activated?
- the 2ndary messengers
- overlapping 2ndary messengers can alter and fine tune responses (convergence)
Adenylyl Cyclase
- activated in response to GCPR activation
- converts ATP > cAMP
- activated G protein activated adenylyl cyclase
Cyclic NT phosphodiesterase
- an activated molecule in response to GCPRs
- convert cAMP > AMP
- aids in stopping/desensitizing GPCR signaling
The general scheme for all GCPRs are
- fairly similar
- involve 1st and 2nd messengers
- utilize the GCPR
- activate a G protein
- conversion of GDP > GTP
Beta-adrenergic signaling ptw
- classical GCPR signaling
- activation of the beta-adrenergic R (BAR) leads to production of cAMP
- involved in fight/flight response; leads to metabolism of sugars (for F or F)
Beta-adrenergic signaling ptw steps
- epinephrine binds to the BAR
- BAR activation > activates Gsa
- Gsa goes to activate adenylyl cyclase
- adenlyly cyclase amplifies cAMP production
- cAMP goes to activate PKA
- PKA phosphorylates cellular proteins to induce a cellular response
- cyclic NT phosphodiesterase converts cAMP > AMP to limit the cellular response when the time is right
Adenylyl cyclase is able to amplify cAMP production because
- the activated BAR can activate multiple Gsa proteins
- multiple Gsa proteins go off to activate multiple adenylyl cyclases
Heterotrimeric G-proteins
- g proteins that cycle b/w active and inactive forms
- inactivate w/intrinsic GTPase activity
How does Gsa function as a heterotrimeric G-protein?
- GTPase activity converts GTP > GDP to inactivate the protein
- inactive (Gsa-GDP) reassociates w/the BAR protein
How can sustained epinephrine signaling lead to desensitization?
- epi > BAR activation
- activated Gsa can recruit BARK
- BARK binds to the BAR
- BARK-BAR complex is endocytosed to prevent/desensitize signaling
The production of cAMP molecules can go off and activate PKA molecules via
- allosteric regulation
- activates PKA to allow it to phosphorylate other proteins downstream
How specifically is PKA allosterically regulated by cAMP molecules?
- inactive PKA: tetramer w/two catalytic and two regulatory subunits
- cAMP binds to the catalytic subunits to induce a conformational change
- conformational change allows regulatory subunits to dissociate, leaving active PKA
Summary of the Beta Adrenergic ptw: what is the signal molecule?
epinephrine/norepinephrine
Summary of the Beta Adrenergic ptw: what is the receptor?
the beta-adrenergic receptor
Summary of the Beta Adrenergic ptw: what is the transducer?
- Gsa (the g protein)
Summary of the Beta Adrenergic ptw: what is the effector molecule?
adenylyl cyclase
Summary of the Beta Adrenergic ptw: what is the 2nd messenger molecule?
cAMP
Summary of the Beta Adrenergic ptw: what is the target of the 2nd messenger?
- PKA
How can you attenuate/stop BAR signaling?
- activation of cyclic NT phosphodiesterase (cAMP > AMP)
- decreasing epi lvls
- activation of phosphatases > inactive PKA
- endocytosis of BAR (BARK)
How are GCPRs involved in metabolism?
- primarily through amplification of signaling molecules
- examples: hepatocytes (and insulin signaling
GCPR activation and metabolism in hepatocytes
- breakdown of glycogen > glucose
- active PKA > activates phosphorylase b kinase (phbk)
- phbk > activates glycogen phosphorylase
- glycogen phosphorylase breaks down glycogen > glucose
GCPR activation and metabolism w/insulin signaling
- insulin binds to the insulinR
- insulinR autophosphorylates and acts as a signaling raft
- IRS-1 is recruited to the raft and becomes phosphorylated
- IRS-1 > activates raf
- raf leads to downstream signaling and transcription of genes needed for cell division
What molecules involved in insulin signaling help amplify the initial signal?
- Raf-1
- MEK
- EKR
- MAP kinases > amplify signal cascade
How is insulin signaling attenuated/stopped?
- decrease in insulin lvls
- regulation of signaling targets via ubiquitination
Ras is considered to be what type of molecule?
- a g protein
- contains a P loop that holds cofactors and GTP
- capable of GTP hydrolysis (for inactivation)
- mutations are highly prevalent in cancers
How are GCPRs involved in other diseases?
- cholera and pertusis
- catalyze the transfer of ADP-ribose to G proteins
- prevents G proteins from recting to normal hormone stimuli
How are GCPRs involved in vision signaling?
- activation of GCPRs lead to ion channel openings
- olfaction and gustation work similarly
GCPR vision signaling steps
- light is absorbed and converts cis-retinal to all-trans-retinal
- conversion activated rhodopsin
- rhodopsin catalyzes the replacement of GDP to GTP on transducin (T)
- T then dissociates and becomes Ta-GTP and Tb^2
- Ta-GTP goes to activate molecules to produce cGMP
Summary of the rhodopsin/vision ptw: what is the signal?
production/accumulation of all-trans retinal
Summary of the rhodopsin/vision ptw: what is the receptor?
rhodopsin
Summary of the rhodopsin/vision ptw: what is the transducer (the G-protein)?
transducin
- Ta-GTP and Tby
Summary of the rhodopsin/vision ptw: What is the effector molecule (2ndary messenger)?
cGMP
Summary of the rhodopsin/vision ptw: what is the target of the 2ndary messenger?
Na/Ca2+ channels
How can the rhodopsin ptw be attenuated/stopped?
- reopening of cation channels
- activation of guanylyl cyclase, arrestin, or rhodopsin kinase
FRET
- fluoresence resonance energy transfer
- used to determine the regulation/activation of certain molecules via fluorescent tags
FRET process/how it works
- a fluorescently tagged molecule becomes excited and transfers its energy to an acceptor molecule w/o emission of a photon
- the acceptor (tagged fluorescent) molecule receives the energy and decays
- decay of the acceptor emits a photon of a longer wavelength than the original light and donor energy
What does fluorescence in FRET assays indicate?
- longer wavelength/2ndary color emission: there are protein-protein interactions happening b/w the two tagged proteins
Adhesion receptors (integrins)
- Rs that binds molecules in the ecm and the actin cytoskeleton
- responds to mechanical stress
Integrins are important in signaling
- from the outside in and vice versa
- integrates cellular activity
Types of integrin signaling
- mechanical
- hormonal
- immunological
Receptor guanylyl cyclases
- tmem and intracellular receptors that are enzymes
- ligand binding to the extracellular domain stimulates the conversion of GTP > cGMP
Tmem RGCs tend to bind what types of ligands?
hydrophilic ligands
Intracellular RGCs tend to bind to what types of ligands?
gaseous and hormonal ligands
How can RGCs be desensitized?
- dephosphorylation
- cGMP breakdown
Nitric Oxide signaling
- an example of RGC signaling
- NO binds to GCRs
- activates soluble NO synthase
- produces nitric oxide which relaxes cardiac muscle
Receptor Tyrosine Kinases/Tyrosine Receptor Kinases
- receptors that have endogenous enzyme activity
- or can directly activate an enzyme upon ligand binding
RTKs specifically activate enzymes/become enzymatic by/when
- ligand binds > dimerizes RTKs
- binding induces a conformational change
- leads to phosphorylation of tyrosine residues
- leaves RTK/enzyme active
RTK phosphorylation is more specifically known as
- transautophosphorylation
- the 'arms'/segments phosphorylate the opposite arm (crisscross)
The result of RTK activation is typically
- activation of kinase cascades
- kinase cascade leads to gene expression (expression of TFs)
RTKS and tyrosine-kinase associated receptors are also known as/called
enzyme coupled receptors
What is the conserved structure of RTKs?
- formation of monomers to dimers
- many receptors stay as monomers until ligand binding
The insulinR and the IGF1R are examples of
RTKs
How are the insulinR and IGFR structurally different from other RTKs?
- is a constitutive dimer
- SS covalent bonds hold the dimers subunits together
Types of RTK ligands
- GFs
- developmental signals
- differentiation signals
How is the information from the signaling ligand transferred across the CM when they bind to RTKs?
- conformational changes in the R
- dimerization of the R
- changes in the localization of the R and proteins
- leads to internal signaling based on the specific ligand that binds to its specific R
Dominant negative RTK receptors
- mutant receptors that are able to bind to ligand and dimerize
- dimerization does not result in signaling due to mutant structure
Dominant negative RTK receptors usually have faulty
tyrosine kinase domains
How could an RTK become constitutively active?
- autocrine secretion of the ligand that binds to RTKs
- leads to activation of RTKs and downstream signaling
- called constitutive dimerization
Signaling molecules/protein molecules that are recruited to activated/phosphorylated RTKs contain what regions?
- SH2 domains (sarc proteins)
- Phosphotyrosine domains
SH2 and phosphotyosine domains on recuited signaling proteins recognize
phosphotyrosines on the RTK
SH2 domains/proteins are specific, meaning
- they are specific proteins that recognize specific phosphotyrosines
- recognize specific am ac side chains when they bind to the activated RTK
Note
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