The document discusses receptor desensitization, which is the decrease in response of cells to agonists after continuous stimulation. It can occur via two types: homologous, mediated by the same receptor; or heterologous, mediated by a different receptor. Factors that can cause desensitization include changes to receptors, loss of receptors, exhaustion of mediators, increased drug metabolism, and physiological adaptation. The mechanism involves phosphorylation of receptors and binding of arrestins, which uncouple the receptor from G proteins and promote internalization. β-arrestins play a key role in desensitization and can also translocate to the nucleus to influence transcription.

1. RECEPTOR DESENSITIZATION Varun m. mehta Department of pharmacology M.Pharm-I (2008-09)

2. CONTENTS Definition Types of desensitization Factors responsible for desensitization Mechanism of desensitization Role of β -arrestin in desensitization Role of desensitization in disease Study techniques of desensitization & instrument

3. RECEPTOR DESENSITIZATION What is receptor Desensitization? • The rapid signal attenuation in response to stimulation of cells by agonists. • In other words decrease in the response of a drug due to frequent administrations. • Continuous stimulation of cells with agonists generally results in a state of desensitization . • also referred to as adaptation, refractoriness, or down-regulation . • The term tolerance is conventionally used to describe more gradual decrease in response of drug which takes days or weeks to develop.

4. -> After exposure to an agonist, the initially maximum response is seen. -> Now if we continue to administer agonist it shows decrease in response due to desensitization of receptors. -> Removal of the drug for a more extended period allows the cell to recover its capacity to respond.

5. Types of Desensitization • Two types 1. Homologous desensitization:- It is mediated by agonist- induced activation of the same receptor. Here agonist desensitizes target cells only to itself. It allows the explanation of complex biological phenomenon such as opioid dependence and tolerence. 2. Heterologous desensitization:- It is caused by activation of a different receptors or through pathway that is common for many receptors. Here one ligand desensitizes target cells to another ligands. GOODMAN & GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS - 11th Ed. (2006) : http://books.google.co.in/books?id=6CL6b3FRoiUC&pg=PA134&lpg=PA134&dq=Receptor+desensitization+%2B+disease&source=web&ots=cMSywmUuHr&sig=cXrOfq8sZZAd13jGTTA7lEFEi6w&hl=en&sa=X&oi=book_result&resnum=6&ct=result#PPA134,M1

6. -> When agonist is administered continuously,desensitization of the receptor results ; when agonist falls below a certain threshold,the system again becomes sensitive. -> E.G. Think of whathappens to your visual transduction system when you walk from bright sunlight into a darkened room or from darkness into the light. Lehninger principles of biochemistry 4th Edition pg.427

7. Five ways by which target cells can become desensitized to a signal molecule. http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mboc4.figgrp.2792

8. Many factors can give rise to phenomenon of desensitization. 1. Change in receptors 2. Loss of receptors 3. Exhaustion of mediators 4. Increased metabolic degradation 5. Physiological adaptation

9. 1. Change in receptors -> desensitisation is often rapid and pronounced with ionic receptors. -> At the neuromuscular junction, the desensitised state is caused by conformational change in the receptor, which will result in the tight binding of the agonist molecule without the opening of the ionic channel. -> Phosphorylation of intracellular regions of the receptor protein is a second, slower mechanism by which ion channels become desensitised . Acetylcholine (ACh) at the frog motor endplate. -> Initially brief depolarisations (upward deflections) are produced by short pulses of ACh delivered from a micropipette. -> A long pulse (horizontal line) causes the response to decline, which shows the ionic receptor desensitisation, and it recovers with a similar time course. In Nut Shell Conformational change in receptor leads to receptor desensitisation.

10. 2.LOSS OF RECEPTORS -> Prolonged exposure to agonists often results in a gradual decrease in the number of receptors expressed on the cell surface , as a result of internalisation of the receptors. -> β-Adrenoceptors of rat glioma cells in tissue culture. Isoprenaline (1μmol/l) was added at time zero, the response and β-adrenoceptor density measured at intervals. During the early phase, the response (blue line) declines with no change in receptor density (red line). Later, the response declines further concomitantly with disappearance of receptors from the membrane by internalisation. The green and orange lines show the recovery of the response and receptor density after the isoprenaline is washed out. In Nut Shell Loss of receptor due to intenalisation lead to receptor desensitisation.

11. 3.EXHAUSTION OF MEDIATORS • In some cases, desensitisation is associated with depletion of an essential intermediate substance. • E.g. Drugs such as amphetamine, which acts by releasing amines from nerve terminals, & show marked tachyphylaxis because the amine stores become depleted . In Nut Shell Depletion of an essential mediators lead to receptor desensitisation.

12. 4.ALTERED DRUG METABOLISM • Tolerance to some drugs, for example barbiturates and ethanol , developes because of repeated administration of the same dose . • Which will produces lower plasma concentration of other drugs as a result of increased metabolic degradation due to enzyme induction. In Nut Shell Increased metabolic degradation of drugs lead to receptor desensitisation.

13. 5.PHYSIOLOGICAL ADAPTATION • Diminution of a drug's effect may occur because it is nullified by a homeostatic response. • For example, the blood pressure-lowering effect of thiazide diuretics is limited because of a gradual activation of the renin-angiotensin system. In Nut Shell Drug effect may be nullified by homeostatic mechanism which may lead to receptor desensitisation.

14. Desensitization can be the result of 1. temporary inaccessibility of the receptor to agonist. 2. or the result of fewer receptors synthesized and available at the cell surface. • Down-regulation of receptor best describes point 2 . • E.g. Agonist stimulation of GPCRs initially produces rapid response but subsequent stimulation by agonist lead to desensitization of the receptor.

15. Mainly receptors can be desensitized in three different ways. 1. Agonist-mediated endocytosis. 2. Anti-receptor antibodies cause relatively slow endocytosis of the receptors. 3. Stress induced receptor desensitization. http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=eurekah.section.59454

16. General Mechanism of Agonist mediated Desensitization Phosphorylation of the receptor by specific GPCR kinases (GRKs) plays a key role in triggering rapid desensitization. 1. Phosphorylation of agonist-occupied GPCRs by GRKs facilitates the binding of cytosolic proteins termed arrestins to the receptor. 2. This results in uncoupling of G protein from the receptor. 3. Now β-arrestins recruit proteins such as PDE4 (which limits cyclic AMP signaling), and others such as clathrin and β 2- adaptin , this all will promot sequestration of receptor from the membrane (internalization).

17. Steps involved in Agonist-induced internalization of GPCRs. 1. Agonist activation of many GPCRs results in receptor phosphorylation by GPCR-specific kinases (GRK). 2. The phosphorylated GPCR recruits β-arrestin , which initiates receptor targeting to clathrin-coated pits. 3. Endosomal acidification permits dissociation of the agonist. 4. The GPCR is dephosphorylated by a G-protein-coupled receptor specific phosphatase (GRP). 5. Internalized receptors can recycle to the cell surface or are degraded in lysosomes.

18. Encyclopedic Reference of Molecular Pharmacology pg. no. 913

19. Another example showing desensitization of β -receptor due to continuous exposure to epinephrine.

20. Desensitization of the β –adrenergic receptor in the continued presence of epinephrine. This process is mediated by two proteins: - Β - adrenergic protein kinase (ARK) & β -arrestin (arr ; arrestin 2). Lehninger principles of biochemistry 4th Edition pg.440

21. Anti receptor antibody(ARAs) • ARAs are pathogenically linked to endocrine disorders, either ↑ or ↓ hormonal activity. • They are directed against receptors for corticotropin, H 2 –histamine, parathyroid, islet of β cells, insulin, thyrotropin–TSH, gastrin, and FSH http://medical-dictionary.thefreedictionary.com/anti-receptor+antibody

22. Stress induced desensitization • These include osmotic stress • stimulation with the tumour necrosis factor (TNFs) and • irradiation by ultraviolet (UV) light. • E.g. acute psychological stress can produce adrenergic receptor densensitization,possibly through increased levels of circulating norepinephrine.

23. Continued …….. • Researchers studied 30 first year medical students during final examination week and one month earlier. Platelet a2 receptor binding was measured using 3H-yohimbine. • During final examination week, platelet α 2-receptor binding affinity was significantly reduced, while levels of plasma catecholamines and anxiety were significantly increased, compared with the earlier period. • This study shows that stress can also play a role in induction of the desensitization. http://www.psychosomaticmedicine.org/cgi/reprint/52/6/624.pdf

24. β -arrestins β -arrestin 1 and β -arrestin 2 are well known negative regulators of G-protein-coupled receptor (GPCR)signaling. 1) They promotes GPCR internalization, thus causing desensitization. or 2) They also function as scaffold proteins that interact with several cytoplasmic proteins. 3) Recent work has also revealed that, in response to activation of certain GPCRs , β -arrestins translocate from the cytoplasm to the nucleus and associate with various transcription cofactors such as p300 and cAMP-response element-binding protein (CREB) to promote transcription. http://jcs.biologists.org/cgi/reprint/120/2/213 Journal of Cell Science

25. Domain Structure of β -arrestin β - arrestins contain two major domains: an N domain and a C domain. 2) The N domain (1-185) is indispensable for the nuclear localization of both arrestins 3) Nuclear export signals (NES) is located at the C-terminus of β -arrestin 2. C-terminus allows to initiate clathrin-dependent endocytosis. 5) The IP6-binding site resides in the C domain (233-251). http://jcs.biologists.org/cgi/reprint/120/2/213 Journal of Cell Science

26. DISEASE RELATED WITH GRK2 EXPRESSION (1) Role of GRK2 in cardiac failure Abnormalities in GPCR receptor signaling pathways results in severe symptoms of heart failure. • animal models of Heart disease shows upregulation of GRK2 expression. • Prooven by experiments: GRK2 over expression. GRK2 inhibition.

27. Continued…… • GRK2 over expression: Over expression resulted in reduced cardiac contractility. • GRK2 inhibition : GRK2 inhibiting peptide quenches the ßγ subunits and inhibits GRK2 activity which results in reduction of hypertrophy & improved β -adrenergic signaling and increased survival.

28. (2) Role of GRK2 in Hypertension • Altered activity of adrenergic, endothelin & angiotensin II receptors in vascular smooth muscle cells has been implicated in hypertension. • Increased expression of GRK2 in epithelial cells inhibits the endothelial nitric oxide synthase activity. • This will lead to decrease in NO production and ultimately cause hypertension.

29. Conclusion • In nut-shell GRK2 upregulation has roles in cardiac failure and hypertension. • So the agents which down regulate the GRK2 may be potential target for the treatment of hypertension.

30. How receptor internalization can be studied? • Receptor internalization can be studied by: - Transfluor Technology - Imaging on live / fixed cells • Tagged receptors • Fluorescent ligands -High Throughput Screening

31. (1) Transfluor Technology Cell-Based Fluorescence Technology for GPCRs Transfluor technology is used for detecting and quantifying GPCR desensitization and internalization. Method :- Here fluorescent is label to beta-arrestin & the location of the receptor-arrestin complex is monitored. Figure 1. Left: unstimulated cells. (1) Activation of the receptor induces, a mass movement of the fluorescence to the cell membrane & forms pits(pits, center), and within few minutes endocytic vesicles are formed(right). http://www.moleculardevices.com/pages/reagents/transfluor.html

32. Video recording of Transfluor Technology

33. live-imaging of tagged adenosine receptors. 0 minutes (3) 24 min. (1) 0 minutes (2) 12 min. (3) 24 min. (4) 36 min. Department of Phsyiology & Pharmacology Receptor Biology & SignalingKarolinska Institutet, Stockholm

34. imaging of endogeneous adenosine receptors internalization Department of Phsyiology & Pharmacology Receptor Biology & SignalingKarolinska Institutet, Stockholm

35. High-Throughput Screening • Opera Imaging Reader APPLICATIONS:- Use to study (1) Ligand binding, (2) Receptor activation and desensitization,

36. REFERENCES -> Rang & Dale’s pharmacology 6 th edition published by Elsevier. -> Goodman & Gilman‘s The Pharmacological Basis of Therapeutics - 11th Ed. (2006) : -> Lehninger principles of biochemistry 4th Edition -> Encyclopedic Reference of Molecular Pharmacology -> Journal of Cell Science -> http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid= mboc4.figgrp.2792 -> http://jcs.biologists.org/cgi/reprint/120/2/213 -> http://www.moleculardevices.com/pages/reagents/transfluor_presentations.html