G protein-coupled receptors

cAMP vs. Calcium termination

-cAMP broken down by enzymes to AMP & removed from cell -calcium cant so it must be moved somewhere else or absorbed -drugs have diff consequences with these 2 factors

Termination of cAMP signaling cascade

-cAMP level: PDE (phosphodiesterase) -protein level: phosphatase

Gsa G-proteins are sensitive to

-cholera toxin -only binds to active form of Gs alpha -cholera toxin's covalent modification of Ga prevents GTP hydrolysis -causing persistent signaling (timer to be stuck on) -Cholera toxin (CTX) ADP-ribosylates an arginine residue on Gsa G-protein subunit. (GTP can no longer come off) -result: elevated cAMP levels.Receptor never stops signaling bc Gsa stays bound to GTP.

GPCR is held together by

-cholesterol molecules -alpha helix loop -cryztalized structures w ligands

partial agonist

has receptor binding affinity andless intrinsic activity (half affinity)

antagonist

has receptor binding affinity but no intrinsic activity -zero efficacy

agonist

has receptor binding affinity plus intrinsic activity -positive efficacy

Heterotrimeric G-proteins

-3 parts to it, encoded by 3 genes, 3 polypeptides G-(alpha (only binds to GTP), beta, and gamma). -Can bind to GTP or GDP. -Ga & Gy are attached to the plasma membrane via lipid anchors

Inverse agonist

-An agent which binds to the same receptor binding-site as an agonist for that receptor but exerts the opposite pharmacological effect" -Difference from Antagonist:Antagonist binds to the receptor, but does not reduce basal activity -Inverse agonists are effective against certain types of receptors (e.g. certain histamine receptors and GABA receptors) which have constitutive activity -negative efficacy

Pathways that regulate calcium levels

-Calcium as 2nd Messenger: powerful & prolific 1. kick it out of cells 2. move excess ca to other parts of cells like ER, mitochondria, away from cytoplasm. 3. Absorbed by proteins (mops)

Pharmacological Antagonists

-Competitive antagonist -signature is diff potency! but efficacy remains the same. -Bind to the same site as the endogenous ligand or agonist. -Their presence produces a right-ward shift in both the binding and dose-response curves -No change in Emax or Bmax. -propanolol: a competitive ßadrenergic receptor antagonist -need more of agonist to overcome competitive antagonist

ex of Irreversible antagonists

-EEDQ (non-selective modification of carboxyl groups) -N-ethylmalemide (NEM) or other sulfhydryl or alkylating agents (non-selective). -Antibodies -Molecular modifications (mutation)

Who researched GPCR?

-Ehrlich (theory of receptors) -Lefkowitzs (cristilize structure of GPCR) -Kobilka (cristilize structure of GPCR) -Gilman (discovered GPCR) -Rodbell (discovered GPCR)

A high throughput assay for measuring calcium levels

-Fluo-3AM: cell permeable, cleaved by esterase to Fluo-3 -Fluo-3: Increased fluorescence when bound to calcium -using Fluo-3AM, hydrophobic molecule goes through the membrane, after it crosses esterases cleave AM and left w Fluo-3. More hydrophilic and gets trapped within the cell. can bind ca, when it binds ca and u excite it w a certain wavelength of light, it emits a flourescence you can measure. useful method

What terminates GPCR Signaling Pathway?

-G alpha has the GTPase activity. -GTPase activity will terminate the convo w the receptor. -terminates when GTP converts to GDP. gamma P gets cleaved and convo stops. -as long as ligand is present this is whats going to happen.

3 components of GPCR signal transduction pathway

-GPCR receptor -Heterotrimeric G protein -Signaling effector (who receives the signal).

Key GPCR Signaling Pathways

-GPCR-Adenylate Cyclase Signaling Pathway -GPCR-Phospholipase C-b Signaling Pathway -GPCR-Phospholipase A2 Signaling Pathway -GPCR-bgamma Subunit Mediated Signaling Pathway

A high throughput assay for measuring G-protein activation

-GTP-Eu: GTP labeled with Europium -works in same was as 35SGTP but has chlorofyl that can be excited at diff wavelengths that can be quantified -cleaner, quicker way, most used now -A lanthanide chelate -Long decay time of fluorescence -Large Stoke's shift (difference between the excitation and emission wavelengths)

Features of G-protein Subunits:

-Ga has GDP/GTP binding pocket. -Ga has ras-like GTPase domain (converts GTP to GDP). -Gy interacts with Gb but not Ga -Gy-Gb forms a stable dimer with coiled-coil N-terminal interactions. -Ga carboxyl tail interacts with GPCR (important in conformational changes) -Ga subunits are myristolated and/or palmitoylated (lipid modified). -Gy subunits are prenylated.

GPCR-bg Subunit Mediated Signaling Pathway

-Gi coupled protein when activated it releases the beta-gamma that bind to the K channel called Gert. -released beta gamma releases K ion (efflux). making the cell more negative. -Important pathway in neuronal regulation and in brain

GPCR-Phospholipase C-b Signaling Pathway

-Gq/ G11 affects phospholipase C, can cleave a molecule present in cell membrane (lipase). -breaks it down to IPS and diacylglycerol. -IP3 is a ligand for intracellular receptor thats present on ER. -IP3 receptor and its a ligand gated channels. -When IP3 bind to receptor it releases calcium. -ca then floods the cells. -2nd messenger is IP3 and the 3rd messenger is ca. cal activates kinase c.

Heterotrimeric G-proteins: G-alpha

-Gs: stimulates adenylyl cyclase. -Gi/o: inhibits adenylyl cyclase and regulates ion channels. -Gq/11: activates phospholipase Cb. -G12/13: activates Na+/H+exchanger pathway.

antagonist ligand binding sites

-Human NK-1 (substance P) receptor -bind closer to cytosol of 7 transmembrane

Using GTPg35S to study GPCR Structure-Function

-Isolate cell/tissue membrane (with receptor & associated proteins) -incubate with ligand and GTPg35S -Filter through membrane & wash -Measure the radioactivity retained on the membrane -obtain sygmoidal curve -determine potency and efficacy -potency shifts to right bc binding affinity change bc of mutation & drug is not binding as well to receptor (signal is less potent) -downside: radioactive label

Disadvantages of irreversible inhibitors

-It is irreversible and effects cannot be reversed by an agonist. -e.g. Phenoxybenzamine, an irreversible α-adreno receptor antagonist used to control hypertension

Novel Concept: Functional Selectivity

-Ligand-induced Differential Signaling ( LIDS) -Agonists exhibiting signaling bias -receptor gives you 3 different responses. bias can have a drug that would only activate A or B or C and not A then B then C. -can find drugs that only activate therapeutic and not gert (K+) w its side effects. -looking to activate 1 and not the other. -complications in drug screening but a lot of promise in the future.

Radioligand binding method: Direct binding assay

-Measures direct interaction of a radioligand with a receptor (how fast it binds & comes off). -Determines kinetic and equilibrium properties. -Provides estimates of receptor density (how much of receptor is present). -Useful for estimating appropriate range of ligand concentration in functional signaling assays.

Radioligand binding method: Indirect Binding or Competition Assay

-Measures the inhibition of the binding of a radioligand by an unlabeled ligand. -More commonly used. less expensive -Useful for studying new ligands. -studying ligands that are not suitable radioligands bc they are too lipophilic or bc the receptor affinity for these compound is too low.

Proteins that modulate G-protein function

-The intrinsic GTPase activity of Ga subunit is low. -Regulators of G-protein Signaling (RGS).

Two types of GPCR-adenylate cyclase signaling pathways

-The stimulatory pathway involves Ga s (stimulates cAMP) -The inhibitory pathway involves Ga i/o (inhibits cAMP)

Scatchard transformation

-Y axis is bound/ free (total radioligand-bound) -X-axis Bound (pmol/mg protein) -Straight lines are easier to interpret -Particularly useful if the receptor has different affinity states

Regulators of G-protein Signaling (RGS).

-a large family of proteins from the G-proteins that regulate The GTPase activity and the association and dissociation of GTP/GDP -terminate both signaling process of Ga and Gb-y

GCPR

-aka metabotropic receptors -7 transmembrane or heptahelical receptors present in cell surface membrane -encoded by largest gene family in most animals -involved in neurotransmission and every aspect of life -ancient receptor involved in many functions -30% of drugs use ligands for GPCR ->50% target GPCRs and its signaling pathways

effector proteins leads to changes in

-altered metabolism -altered gene expression -altered cell shape or movement

Partial agonists

-balances low & excessive activity -Some agonists never elicit a maximal response (compared to the endogenous agonist) even when all receptors are full -EC50 are remarkably close to full agonists. -Similar potency, but lower efficacy: Intrinsic activity = 0~1 -high receptor binding affinity for the receptor. -partial functional activity. -acts as both agonist and antagonist.

Convertion of GTP to GDP

-by G-alpha -loosing the outermost phosphate (gamma P)

How are GPCR's activated by ligands?

-conformational changes -using the structure of the GPCR bovine rhodopsin w retinal bound, (conformational change in rhodopsin from cis to trans) that activates signal cascade. -ligand alter receptor conformation activates G-proteins.

Receptor occupancy and biological response

-dont confuse w binding -measuring biological response -can have a drug full agonist for cAMP but have no effect (neutral antagonist) in the gert part -full agonist -partial agonist -neutral antagonist -partial inverse agonist -full inverse agonist

Advantages of Irreversible antagonists

-duration of action of such an inhibitor is relatively independent of its rate of elimination and more dependent on the rate of turnover of receptor molecules. -Once the receptor is occupied by antagonist, the inhibitor need no longer be resent in unbound form to inhibit the effects of an agonist.

phosphatase

-enzyme used to remove phosphorylation of protein induced by catalytic subunit of PKA from cAMP -brings protein back to nonphosphorylated form

Radioligand binding methods

-estimating # of density of receptors in tissues -Discriminating multiple classes of receptors and determining relative proportions (based on ligands you see) -Determining receptor properties such as binding affinities

3 families of GPCR

-family A: rhodopsin/ beta2 adrenergic receptor-like (majority) -family B: glucagon/VIP/ calcitonin receptor-like -Family C: metabotropic NT/ ca receptors

transmembrane regions have

-globular structure w various pockets formed within structure. -helixes all have different angles. -highly dynamic molecules. -the angles change, the shape changes, but still retain their globular structure. -within restrain theres dynamic movement w ligand and activation of receptor.

agonist ligand binding sites

-human B2 adrenergic receptor -binds catecholamines in middle of 7 transmembrane

GPCR-Phospholipase A2 Signaling Pathway

-important in clinical studies -similar to phospholipase c but u have A2 and it releases arachidonic acid (2nd messenger) which can affect oxygenase enzymes. -important bc aspirin (cyclooxygenase) and the singulair (leukotrienes) inhibit oxygenase enzymes.

determine total binding

-initially a lot of receptors available, at low con, more possibility of binding -then saturate receptors present and doesnt get initial response (exponential increase). -receptor system always saturates -doenst tell you anything about specific binding thats what your interested is.

Irreversible antagonists

-making more receptors gets around it -Binds in an irreversible manner, usually by covalent modification of the receptor. -Effectively and practically lowers the number of receptors capable of binding an agonist. -Adding more agonist does not rescue response. -New receptor synthesis is required to restore response.

GPCR-Adenylate Cyclase Signaling Pathway

-most common pathway -adenulate cyclase enzyme activated by Gsa -generates 2nd messenger cAMP from ATP -cAMP activates kinase (PKA) that modifies other proteins -signaling cascade that affect several functions

Gi/oaG-proteins are sensitive to

-pertussis toxin -stops cycle by preventing signaling -its covalent modification of Ga prevents GDP (inactive form) dissociation -causes timer to be stuck off -Pertussis toxin (PTX) ADP-ribosylates a cysteine residue (position -4 from carboxyl terminus end) on Gi/oaG-protein subunits. -used to distinguish between Gsa and Gsi/o (stops signaling its Gsi/o)

Hormesis

-pharmacological exp need to have a dose response in which more is not better. -if conc is given carefully biological response can go away in higher conc (hormesis) happens more than u think.

Other Termination of signaling-common mechanisms

-receptor sequestion -receptor down regulation -receptor activation -inactivation of signaling protein -production of inhibitory protein

Allosteric Modulation

-relevant for new drugs in the market -modulates things 2 ways 1. positive (PAM): shifts potency & increase agonistic effect 2. negative (NAM): reduces efficacy like non competitive antagonist but more gentle. Partially decreasing the efficacy. Important in modulating NT effects that u dont want to completely wipe out

Non-competitive antagonist

-shift in efficacy but not in potency -Does not prevent formation of the DR complex, but impairs the conformation change which riggers a response. -Pseudo-irreversible -Emax and Bmax are reduced but EC50 remains the same for the unaffected receptors.

Temporal nature of signaling

-signaling can be fast (micro secs) or it can be very slow -GCPR signaling is slow (mins) -ligand gated channels are much faster -timing is critical -how long does it last & how quickly it terminates

Contact-dependent

-signaling that target receptor on target cell -have to be in physical contact to occur

How does signaling occur?

-starts w signaling molecule signaling by cell -point of pharmacology is to make a drug that will compete with this signaling molecule & receptor binding -competition between foreign molecule and the endogenous molecule present. -complementing it or antagonizing it -substrate for competition, once bound to ligand you have a signal tranduction process that amplifies signal.

Direct binding assay results:

-subtract the data of total binding and specific binding and you get specific binding. -non linear -initial rapid binding then it saturates.

aspirin affects what enzyme in the GPCR phospholipase A2 enzyme?

-target is cyclooxygenase -effects prostaglandins & thromboxanes which are inflamatory reagents.

singulair affects what enzyme in the GPCR phospholipase A2 enzyme?

-target is leukotrienes -prevent asthma, histamine rxn

Toxin Sensitivity of Two Major Classes of Heterotrimeric G-proteins

-used to determine between the 2 major classes of heterotrimeric G-proteins -Gsa G-proteins are sensitive to cholera toxin. -Gi/oa G-proteins are sensitive to pertussis toxin.

PDE (phosphodiesterase)

-used to reduce cAMP levels -breaks cAMP recycles it back to ATP

G-protein receptor kinase/b-arrestin mediated termination of signaling

-used to terminate signaling after activation has been too long (even if ligand is present) -G-protein kinase (GRK) phosphorylates the receptor (adding P on aa receptor) -results is arresting binds and turns of the whole signaling -discovered using anergic receptor signaling (saw many side affects on cardiac)

Non-hydrolyzable GTP analogs

-uses assay to quantify how much G protein was activated by stabilizing an intermediate step -GTPgS and Gpp(NH)p. (more common & radioactive) -[35S] GTPgS is commonly used to study functional activation of GPCR by ligands. (added instead of GTP & cant be hydrolyzed). -playing w gamma phosphate (removing an O and substituying it w a sulfur atom).

Partial Agonists-Ideal Drugs for Treating Schizophrenia?

1. Act as functional antagonist: improving + symptoms -Increased dopaminergic transmission in mesolimbic pathway-positive symptoms. 2. Act as a functional agonist: improving - symptoms -Decreased dopaminergic activity in mesocortical pathway-negative symptoms. 3. Incomplete blockade of dopamine receptors in the mesostriatal and tuberoinfundibular pathway-decreasing EPS and sexual side effects.

Receptor antagonists

1. Chemical Antagonists: One drug may antagonize the action of a second by binding to and inactivating the second drug -protamine binds heparin 2. Physiological Antagonists: take advantage of physiologic antagonism between endogenous regulatory pathways. -insulin vs. glucocorticoids 3. Pharmacological Antagonists: competing for the same receptor, block the ability of agonists to bind to receptor or may bind to another site that blocks the action of the agonist.

GPCR Signaling Pathway

1. conformational change between receptor & ligand 2. info is conveyed to Galpha, exchange of GDP for GTP 3. get removal of GDP, GTP comes into the binding pocket of the G alpha 4. GTP interacts w G alpha, activated G protein complex. 5. this activated complex talk to any signaling affectors. 6. terminates when GTP converts to GDP. gamma P gets cleaved and convo stops. -cycle starts all over again.

tools to measure cAMP levels

1. serotonin 2. Competition assay: Labeled cAMP is displaced by the unlabeled cAMP generated by receptor activation. The decrease in the FRET signal provides a measure of cAMP produced by receptor activation. -FRET: fluorescence resonance energy transfer 3. Protein Kinase A (PKA): The Enzyme (protein) activated by cAMP -PKA has regulatory subunit and catalytic subunit (places P on substrate) -transcription factors such as CREB (measuring how much CREB was phosphorylated).

Family A GPCR

7 transmembrane structure: cylindrical structure is an alpha helical structure 2 features that make it part of family A 1. DRY aa (tyrosine asparagine & aspartate) sequence is key characteristic in the 2nd intracellular loop. 2. palmintoylated cysteine in the carboxy tail (cysteine residue modified by lipid) hydrophobic modification allows cysteine to interact w the transmembrane region and thats a regulatory step for many of the GCPR function

4 types of signaling

A. contact-dependent B. paracrine C. synaptic D. endocrine

G-alpha is inactivated when

GDP is bound to its pocket

G-alpha is activated when

GTP is bound to its pocket

Examples of GPCR FDA approved

Pepcid, allegra, claritin, zyrtex, plavix, imitrex, abilify, risperadal, mirapex -huge market

how do u determine specific binding?

determines specific binding: using a cold non radioactive ligand than binds to the receptor and prevents the hot ligand from binding to the specific sites

Partial agonist drug used in Schizophrenia?

ex. Aripiprazole (Abilify)]

paracrine

signaling cell secreting a ligand that binds to receptors in other cells & activates a signaling cascades

endocrine

signaling contact w hormone, goes into bloodstream reaches various issues located else where

synaptic

special signaling in brain & neuromuscularjunction tight small spaces (synapse has receptors)

How do you determine binding properties?

using Radioligand binding methods

binding sites that are critical for agonist are

very diff than binding sites for antagonist -2 diff structural requirements -using mutagenesis studies. (mutated aa no longer binds)

In the absence of full agonist, a partial agonist

will bind the receptor and elicit a partial functional response. -improve biological response

In the presence of a full agonist, a partial agonist

will compete for binding to receptor and elicit smaller response from the full agonist. -excess causing problem so tone it down