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Mechanisms in Desensitization:

Mechanisms in Desensitization

Variation in drug responsiveness:

Variation in drug responsiveness Idiosyncratic drug response – unusual, one that is infrequently observed in most patients Caused by: Genetic differences in metabolism Immunologic mechanism (allergy) Hyporeactive – intensity of effect is decreased Hyperreactive – intensity of effect is increased Hypersensitivity – allergic or other immunologic response to drugs resulting from previous sensitizing exposure Tolerance &Tachyphylaxis are also there which make receptor regulation

Some important terms:

Some important terms Desensitization Tachyphylaxis Refractoriness Fade/waning of response Resistance Tolerance down regulation


Description Fade/waning of response: same Refractoriness/unresponsiveness : Voltage gated ion channels require resting phase before getting activated. During this recovery phase they are called refractory. Resistance : microbes Tolerance: responsiveness usually decreases as a consequence of continued drug administration (days to weeks for recovery) Need greater doses of a drug to produce original degree of effect as time progresses or need to substitute different drug


Description Tachyphylaxis – responsiveness diminishes rapidly after administration of a drug (the first few doses), very rapid tolerance Down regulation- decrease in no of receptors down regulation is the part of desensitization which include 1.Endocytosis (internalization) 2. Receptor Trafficking or 3. Receptor Recycling (Recycling)

Down-regulation :

Down-regulation Increases receptor internalization and degradation Slower onset and more prolonged effect than desensitization Occurs over hours or days Is an agonist-induced decrease in the total # of cell-surface receptors Intensity and duration of action of EGF, PDGF, and other agents that act via tyrosine kinase receptor are limited because of this process Cells responsiveness to ligand is correspondingly diminished


Description Desensitization : Effect of a drug often diminishes when given continuously or repeatedly commonly called desensitization (sec to minutes for recovery) caused by: Change in receptor Loss of receptor ( downregulation ) Exhaustion of mediators Active efflux of drug Enhanced metabolism Compensatory physiological mechanisms


Desensitization Receptor-mediated responses to drugs and hormonal agonists often desensitize with time. After reaching an initial high response, the effect diminishes over seconds or minutes even in the continued presence of the agonist. This desensitization is usually reversible. Thus several minutes after removal of the agonist, a second exposure to agonist results in a similar response. molecular basis of desensitization is unknown.

Drug Desensitization:

Drug Desensitization Receptor Mediated loss of receptor function reduction of receptor number Non-Receptor Mediated reduction of receptor-coupled signaling components reduction of drug concentration physiological adaptation

Receptor Mediated Drug Desensitization:

Receptor Mediated Drug Desensitization 1 . Loss of Receptor Function rapid desensitization due to change in receptor conformation usually due to feedback of cellular effects of agonist E. g phosphorylation of specific amino acids in G-protein coupled receptors blocks coupling to G-proteins

Receptor Mediated Drug Desensitization:

Receptor Mediated Drug Desensitization 2. Reduction of Receptor Number slower , long-term desensitization due to change in receptor number usually due to feedback of cellular effects of agonist E. g phosphorylation of specific amino acids in G-protein coupled receptors causes removal from cell surface

Non-Receptor Mediated Desensitization:

Non-Receptor Mediated Desensitization 1. Reduction of Receptor-Coupled Signaling Components depletion of signaling molecules required for biological response Example: prolonged stimulation of G-protein coupled receptors can lead to depletion of intracellular secondary messengers 2. Increased Metabolic Degradation increase in the rate of metabolism and/or elimination of drug lowers plasma drug concentrations Example: barbiturates induce the expression of metabolic enzymes ( cytochrome P450s) that degrade this drug 3. Physiological Adaptation reduction or amelioration of drug effects due to opposing homeostatic response very few well characterized mechanisms **all of these receptor and non-receptor dependent factors can also contribute to interindividual differences in drug response**

Types of desensitization:

Types of desensitization Homologous: stimulus 2 = stimulus 1 Dependent on receptor phosphorylation but independent of second messengers. Heterologous : stimulus 2 ≠ stimulus 1 Dependent on receptor phosphorylation and mediated by second messengers.

Graphical representation Homologous desensitization:

Graphical representation Homologous desensitization Response of drug A & B After chronic exposure Response of drug A & B

Graphical representation Heterologous desensitization:

Graphical representation Heterologous desensitization Response of drug A & B After chronic exposure Response of drug A & B


Comparison Homologous desensitization Loss of sensitivity only to the class of agonist used to desensitise the tissue. E. g β1 receptors in myocardium Heterologous desensitization Stimulation by one agonist which leads to a broad pattern of unresponsiveness to further stimulation by a variety of other agonists. E. g inhibition of chemotaxis mediated by   µ and δ but not қ G opiate receptors. inhibition of chemokine -induced chemotaxis by opiates is due to heterologous desensitization through phosphorylation of chemokine receptors.

Agonist-promoted GPCR phosphorylation is an essential component of desensitization:

Agonist-promoted GPCR phosphorylation is an essential component of desensitization GPCRs become phosphorylated upon agonist stimulation. Phosphorylation may be catalyzed by 2 nd messenger dependent kinases and/or by G protein-coupled receptor kinases (GRKs). The GRK catalyzed phosphorylation is thought to be responsible for homologous desensitization. Phosphorylated GPCRs display a reduction in Emax and/or and increase in the EC50 when activated by agonists in reconstituted systems. Mutation or removal of the phosphorylation sites of GPCRs retards the onset of desensitization, but desensitization still occurs (Nature 333: 370, 1988). Dominant-negative mutants (or chemical inhibitors) of second messenger-dependent kinases and of GRKs impair phosphorylation and desensitization.

Phosphorylation of GPCR:

Phosphorylation of GPCR GRKs previously called b-ARK (GRK 1 to 7) β- arrestins (1 & 2) Other Protein kinases PKA PKC

Types of GRKs:

Types of GRKs 7 mammalian GRKs - serine/ threonine protein kinases that phosphorylate and regulate agonist-occupied or constitutively active GPCR. 4 sub-groups within the GRK family Visual GRK (only found in retinal cells): GRK subfamily 1: GRK1 ( rhodopsin kinase ) & GRK7 (cone opsin kinase ) 2. Non visual GRK: GRK subfamily 2: GRK2 (b-ARK1) GRK subfamily 3: GRK3 (b-ARK2) GRK subfamily 4: GRK4, GRK5 and GRK6

Desensitization in GPCRs:

Desensitization in GPCRs

Desensitization in GPCRs:

Desensitization in GPCRs Following GPCR activation of G proteins (A), one of the target proteins for the beta/gamma subunit is a kinase called G protein-coupled receptor kinase , GRK (formerly called beta-adrenergic receptor kinase = BARK). As the name suggests, this kinase phosphorylates the receptor (B). The phosphorylated receptor is now in a state that that it can bind the cytosolic protein beta- arrestin (C in figure). The binding of beta- arrestin with the receptor- ligand complex inactivates or desensitizes it so the receptor can no longer activate G proteins. GRK phosphorylation of GPCRs has traditionally been thought of as a sort of internal "negative feedback loop" in GPCR signaling. The activated receptor causes its own inactivation. Following inactivation by beta- arrestin , the arrestin -receptor complex often undergoes endocytosis and subsequently the receptor is degraded by lysosomes .

Essential characteristics of GRKs:

Essential characteristics of GRKs They are S/T kinase and they prefer activated receptors as substrates. Some GRKs are cytosolic but they are translocated to the membrane upon activation (GRK1- GKR3). Some GRKs are peripheral membrane proteins (GRK4 -GRK6). In vitro and in transfecto studies reveal little or no specificity for GRK/GPCR pairs or “consensus” phosphorylation sites. GRK-catalyzed GPCR phosphorylation usually but not always occurs in the C-terminal tail of GPCRs. Known exceptions include : α2AR, m2MR ( phosphorylated in the 3d intracellular loop) FSHR ( phosphorylated in the 1st and 3d intracellular loops )

2. Arrestin family Pharmacol. Rev. 53: 1, 2001:

2. Arrestin family Pharmacol. Rev. 53: 1, 2001 Visual arrestin ( Arrestin 1) Visual arrestin plays a key role in the regulation of rhodopsin signaling in rod photoreceptors Non Visual arrestins ( β arrestins )( Arrestin 2 & 3 ) Both are involved in GPCR regulation Arrestin 2 is the most abundant subtype in mature neurons Non-visual arrestins 2 and 3 recently shown to shuttle between the nucleus and the cytoplasm Arrestin3 with its native nuclear export signalling

β Arrestins : Role:

β Arrestins : Role Role in desensitization β- arrestin -mediated receptor internalization- regulate signal transduction. The internalized GPCR-β- arrestin complex can form a signalosome that activates signaling proteins, such as ERK1/2, p38 MAPK, and JNK. Arrestins act as scaffolds that connect activated GPCR with tyrosine kinase ,NF- κ B & other pathways GRKs and arrestins also interact with non-GPCR. E.g GRKs and arrestins interact with TGF-β, EGF & insulin growth factor receptors. β- arrestin regulate activity of Notch, an important protein in neurogenesis , angiogenesis, and lymphoid development. GRKs and arrestins may directly affect functioning of these non-GPCR or modulate signaling of these receptors indirectly.

GRKs and Arrestins:

GRKs and Arrestins

Role of β arrestin:

Role of β arrestin # class A GPCR e.g β2-adrenoceptor, internalize through a – β arrestin-2-dependent mechanism # class B receptors e.g angiotensin receptor AT1A, have no preference for β - arrestin 2 over β - arrestin 1. # In the case of class A receptors , β - arrestin –GPCR interaction is transient, and β - arrestin does not localize with the GPCR in endosomes . # For class B receptors , the β - arrestin –GPCR interaction is more stable, and the receptor and β - arrestin co-localize in endosomes .

Role of clathrin & PKs:

Role of clathrin & PKs clathrin -coated pits formed dynamin causes fission from plasma membrane and fusion with endosomes . PLD 2 – a widely distributed phospholipid -specific diesterase that hydrolyses phosphatidylcholine to phosphatidic acid and choline and is assumed to play an important function in cell regulation and receptor trafficking Activation of phospholipase D 2 is required for agonist-induced mu- opioid receptor Endocytosis

3. Second messenger-dependent kinases phosphorylates GPCRs:

3. Second messenger-dependent kinases phosphorylates GPCRs Phosphorylation sites may be present in intracellular loops and/or C-terminal tail The β2AR is phosphorylated by PKA at two sites located in the 3 rd intracellular loop and two sites located in the C-terminal tail Initially thought to be important in heterologous desensitization. The PKA mediated phosphorylation of the β2AR is also likely to be involved in switching G protein specificity

GPCR regulation by GRKs and arrestin:

GPCR regulation by GRKs and arrestin ( a ) The classical model of GPCR regulation by GRKs and arrestins The GPCR is activated by agonist (1) leading to G protein coupling (2) and effector modulation. The agonist-occupied GPCR is subsequently phosphorylated by GRK (3), arrestin binds to the phosphorylated GPCR, leading to receptor desensitization (4), internalization (5), dephosphorylation (6), recycling (7) of the GPCR. Particularly with longer agonist treatments, internalized GPCR may also be targeted for downregulation .

GPCR regulation by GRKs, arrestins and PKs:

GPCR regulation by GRKs, arrestins and PKs ( b ) GPCR regulation by second messenger-dependent protein kinases , in this example protein kinase A (PKA) the agonist binds to the GPCR (1) leading to Gs activation (2) and increased levels of cyclic AMP, which activates PKA. The kinase is then able to phosphorylate both agonist occupied (3) and unoccupied (4) GPCRs. This phosphorylation causes desensitization by uncoupling the GPCR from G protein or in the case of the agonist unoccupied receptor by preventing GPCR coupling to G protein. Whether or not GPCR phosphorylation by second messenger-dependent protein kinases leads to arrestin binding or internalization depends upon the particular GPCR subtype in question.

GPCR regulation by GRKs and PKs:

GPCR regulation by GRKs and PKs ( c ) Direct and indirect mechanisms of GPCR regulation by second messenger-dependent protein kinases activated protein kinase C (PKC) can directly phosphorylate and desensitize the GPCR (1), or PKC can phosphorylate and activate GRK2, which consequently has an enhanced ability to phosphorylate the GPCR (2), or PKC can phosphorylate other, as yet unidentified, regulatory proteins, which then effect GPCR desensitization (3).

Regulation of GPCR desensitization by GRK2 and β-arrestins:

Regulation of GPCR desensitization by GRK2 and β -arrestins receptor activation → recruitment of GRK2 by G to the receptor where it is anchored by PIP2 and positioned to phosphorylate the C terminus of the receptor. GRK2 activity enhanced by its phosphorylation by PKA, PKC, and c- Src . GRK2 activity can be reduced by its phosphorylation by Erk1/2 or its binding to calcium/ calmodulin ( CaM ).

Regulation of GPCR desensitization by GRK2 and β-arrestins:

Regulation of GPCR desensitization by GRK2 and β - arrestins GRK2 can also inhibit receptor signaling by sequestering Gq to prevent its coupling to its effectors, such as PLC.

Regulation of GPCR desensitization by GRK2 and β-arrestins:

Regulation of GPCR desensitization by GRK2 and β -arrestins Gs-coupled receptors activate the effector adenylyl cyclase (AC) to generate the second messenger cAMP . After phosphorylation of the receptor by GRK, β - arrestin is recruited to the receptor, translocating PDE4 with it. This places the PDE4 on the membrane, the site of cAMP generation, thus allowing it to efficiently degrade cAMP to AMP and reduce signaling.

Endocytosis actually plays a protective role:

Endocytosis actually plays a protective role in recycling receptors, protecting them from continuous activation/overstimulation, prevent adaptive processes eventually leading to dependence RAVE = relative activity versus endocytosis – Net amount of signal transmitted to the cell as a result of agonist activity and receptor endocytosis . Morphine = high RAVE value, therefore can’t cause endocytosis Endocytosis indeed contributes to functional desensitization of signal transduction by reduction the number of receptors present in the plasma membrane But at the same time Contributes to functional resensitization of signal transduction by promoting dephosphorylation and recycling of receptors to the plasma membrane.

Tachyphylaxis :

Tachyphylaxis Nitroglycerine - drug-free intervals req ’ when administered transdermally Repeated doses of ephedrine – tachyphylaxis . since it is an indirectly acting sympathomimetic amine which will deplete noradrenaline from the nerve terminal. Thus repeated doses result in less noradrenaline being released than the initial dose. Nicotine over the course of a day - the mechanism of this action is unclear.* Hydralazine -if given as a monotherapy for antihypertensive treatment. It is administered with a beta-blocker with or without a diuretic. Dobutamine , a direct-acting beta agonist used in congestive heart failure, also demonstrate tachyphylaxis Desmopressin used in the treatment of type 1 von Willebrand disease is generally given every 12-24 hours in limited numbers due to its tachyphylactic properties. Hormone replacement in menopausal women, oestrogen and progesterone implants can lead to tachyphylaxis

PowerPoint Presentation:

Tachyphylaxis after repeated administration of ephedrine (decrease in its effect on blood pressure ) E = administration of ephedrine

PowerPoint Presentation:

Desensitization of receptors Repeated administration of an agonist (such as epinephrine) over a short time period, results in diminished response of the cell. Following a period of rest, administration of the drug results in a response of the original magnitude. Repeated injection of drug Time Response 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 (according to Lippincott´s Pharmacology, 2006)

GRK mediated desensitization:

GRK mediated desensitization Involvement of GRK5 in Homologous Desensitization of the TSH receptor

Opiod Receptor desensitization:

Opiod Receptor desensitization Series of events occurring after agonist induced activation of opioid receptors (a) Acute signaling: receptor-mediated activation of heterotrimeric G proteins. The triangular agonist could be either an opioid peptide or a drug. The heterotrimeric G protein could be either Gi or Go. The effector pathways could be ion channels, adenylyl cyclase or kinase cascades. (b) Rapid desensitization: phosphorylation and arrestin binding prevent activation of G proteins. (c) Endocytosis : arrestin promoted concentration of receptors in a clathrin coated pit and dynamin dependent formation of endocytic vesicles. (d) Post- endocytic sorting ( i ) Recycling to the plasma membrane termed ‘ resensitization ’. (ii) Trafficking to lysosomes termed ‘down-regulation’