1. Hormone Receptors on the Plasma Membrane Characteristics of Receptors in General Five Groups of Membrane-Bound Receptors The G Protein-Coupled Receptor Superfamily Signal Transduction through Cyclic AMP Signal Transduction through Phospholipase C Role of Calcium Role of Protein Kinase C

2. General Characteristics of Receptors Receptors bind hormones, resulting in a biological response All receptors exhibit general characteristics: - Specific Binding (structural and steric specificity) - High Affinity (at physiological concentrations) - Saturation (limited, finite # of binding sites) - Signal Transduction (early chem event must occur) - Cell Specificity (in accordance with target organ specificity).

3. Specific Binding A receptor will only bind (recognize) a certain hormone, or closely related hormones. LH hCG FSH LH Receptors LHhCG FSH

4. Receptors Have High Affinity In the bloodstream, there are thousands of different peptides. Hormones are present in very small quantities (nanogram or picogram). Receptors must therefore be very sensitive to the presence of a hormone (they must be able to bind the hormone even if it is present in low amounts). Thus, they have high affinity (ability to bind at low hormone concentrations).

5. Analysis of Receptor Binding Sites Na- I + hCG 125 -hCGI 125 TRACER TESTIS Seminiferous Tubules Leydig/Interstitial Cells RT O/N WASH, PBS Centrifuge lactoperoxidase method Count Pellet CPM

6. Receptor must possess structural and steric specificity for a hormone and for its close analogs as well. Receptor must possess structural and steric specificity for a hormone and for its close analogs as well. Receptors are saturable and limited (i.e. there is a finite number of binding sites). Receptors are saturable and limited (i.e. there is a finite number of binding sites). Hormone-receptor binding is cell specific in accordance with target organ specificity. Hormone-receptor binding is cell specific in accordance with target organ specificity. Receptor must possess a high affinity for the hormone at physiological concentrations. Receptor must possess a high affinity for the hormone at physiological concentrations. Once a hormone binds to the receptor, some recognizable early chemical event must occur.Once a hormone binds to the receptor, some recognizable early chemical event must occur. Criteria for hormone-mediated events

7. Affinity: The tenacity by which a drug binds to its receptor. – Discussion: a very lipid soluble drug may have irreversible effects; is this high-affinity or merely a non-specific effect? Intrinsic activity: Relative maximal effect of a drug in a particular tissue preparation when compared to the natural, endogenous ligand. – Full agonist – IA = 1 (*equal to the endogenous ligand) – Antagonist – IA = 0 – Partial agonist – IA = 0~1 (*produces less than the maximal response, but with maximal binding to receptors.) Intrinsic efficacy: a drugs ability to bind a receptor and elicit a functional response – A measure of the formation of a drug-receptor complex. Potency: ability of a drug to cause a measured functional change.

8. Receptors have two major properties: Recognition and Transduction Recognition: The receptor protein must exist in a conformational state that allows for recognition and binding of a compound and must satisfy the following criteria: Saturability – receptors exists in finite numbers. Reversibility – binding must occur non-covalently due to weak intermolecular forces (H-bonding, van der Waal forces). Stereoselectivity – receptors should recognize only one of the naturally occurring optical isomers (+ or -, d or l, or S or R). Agonist specificity – structurally related drugs should bind well, while physically dissimilar compounds should bind poorly. Tissue specificity – binding should occur in tissues known to be sensitive to the endogenous ligand. Binding should occur at physiologically relevant concentrations.

9. The failure of a drug to satisfy any of these conditions indicates non- specific binding to proteins or phospholipids in places like blood or plasma membrane components.

10. Receptors have two major properties: Recognition and Transduction Transduction: The second property of a receptor is that the binding of an agonist must be transduced into some kind of functional response (biological or physiological). Different receptor types are linked to effector systems either directly or through simple or more-complex intermediate signal amplification systems. Some examples are: Ligand-gated ion channels – nicotinic Ach receptors Single-transmembrane receptors – RTKs like insulin or EGF receptors 7-transmembrane GPCRs – opioid receptors Soluble steroid hormones – estrogen receptor

11. Predicting whether a drug will cause a response in a particular tissue Factors involving the equilibrium of a drug at a receptor. Limited diffusion Metabolism Entrapment in proteins, fat, or blood. Response depends of what the receptor is connected to. Effector type Need for any allosteric co-factors – THB on tyrosine hydroxylase. Direct receptor modification – phosphorylation

12. Receptor theory and receptor binding. Must obey the Law of Mass Action and follow basic laws of thermodynamics. Primary assumption – a single ligand is binding to a homogeneous population of receptors NH + 3 COO -

13. k on = # of binding events/time (Rate of association) = [ligand]  [receptor] k on = M -1 min -1 k off = # of dissociation events/time (Rate of dissociation) = [ligand  receptor] k off = min -1 Binding occurs when ligand and receptor collide with the proper orientation and energy. Interaction is reversible. Rate of formation [L] + [R] or dissociation [LR] depends solely on the number of receptors, the concentration of ligand, and the rate constants k on and k off. k on /k 1 [ligand] + [receptor] [ligand  receptor] k off /k 2

14. At equilibrium, the rate of formation equals that of dissociation so that: [L]  [R] k on = [LR] k off K D = k 2 /k 1 = [L][R] [LR] *this ratio is the equilibrium dissociation constant or K D. K D is expressed in molar units (M/L) and expresses the affinity of a drug for a particular receptor. K D is an inverse measure of receptor affinity. K D = [L] which produces 50% receptor occupancy

16. Once bound, ligand and receptor remain bound for a random time interval. The probability of dissociation is the same at any point after association. Once dissociated, ligand and receptor should be unchanged. If either is physically modified, the law of mass action does not apply (receptor phosphorylation) Ligands should be recyclable.

17. Receptor occupancy, activation of target cell responses, kinetics of binding Activation of membrane receptors and target cell responses is proportional to the degree of receptor occupancy.Activation of membrane receptors and target cell responses is proportional to the degree of receptor occupancy. However, the hormone concentration at which half of the receptors is occupied by a ligand (K d ) is often lower than the concentration required to elicit a half- maximal biological response (ED 50 )However, the hormone concentration at which half of the receptors is occupied by a ligand (K d ) is often lower than the concentration required to elicit a half- maximal biological response (ED 50 )

18. Receptor Fractional Occupancy F.O. = [LR]____ = [LR]___ *now substitute the KD equation. [Total Receptor] [R f ] + [LR] [R] = K D [LR]  F.O. = [Ligand] [L] [Ligand] + K D Use the following numbers: [L] = K D = 50% F.O. [L] = 0.5 K D = 30% F.O. [L] = 10x K D = 90%+ F.O. [L] = 0= 0% F.O. 100 50 0 Ligand Concentration Fractional Occupancy

19. Assumptions of the law of mass action. All receptors are equally accessible to ligand. No partial binding occurs; receptors are either free of ligand or bound with ligand. Ligand is nor altered by binding Binding is reversible Different affinity states?????

20. Studies of receptor number and function We can directly measure the number (or density) of receptors in the LR complex. Ligand is radiolabeled ( 125 I, 35 S. or 3 H). Selection of proper radioligand: – Agonist vs. antagonist (sodium insensitive) – Higher affinity for antagonists – Longer to steady state binding Saturation binding curve-occurs at steady state conditions (equilibrium is theoretical only). Demonstrates the importance of saturability for any selective ligand. Provides information on receptor density and ligand affinity and selectivity.

21. Scatchard transformation Y-axis is Bound/Free (total radioligand-bound) X-axis Bound (pmol/mg protein) Straight lines are easier to interpret.

22. The amount of drug bound at any time is solely determined by: – the number of receptors – the concentration of ligand added – the affinity of the drug for its receptor. Binding of drug to receptor is essentially the same as drug to enzyme as defined by the Michelis-Menten Equation.

23. Thus, to reiterate…,Calculating Affinity Take a cell which has the receptor on it (ie, granulosa cells with FSH receptor). Prepare membrane homogenate. Incubate membranes with increasing amounts of labeled hormone. Determine how much binding of hormone occurs at each dose. Dissociation constant (Kd) is dose where 50% of maximal binding occurs. % binding Dose of Hormone 10301003001000 100 50 Kd 0

24. Thus, to reiterate…,Saturation There is a finite limit to the numbers of receptors which can be on a cell. Therefore, there’s a maximum amount of binding which can occur (all receptors are saturated) % binding Dose of Hormone 10301003001000 100 50 0 saturation

25. Biological Response to Ligand Binding A receptor not only binds hormone; there must also be a biological response from the cell (e.g., increased transcription, phosphorylation, etc.) This is also called “signal transduction”. The biological response can result from: - the ligand itself (e.g., Fe, LDL) - the receptor (e.g., increased cyclic AMP, transcription, phosphorylation)

26. Determinants of Biological Response The strength of the response of the cell to the hormone depends upon three factors: 1) the amount of hormone present to bind to the receptors 2) the numbers of receptors on the cell 3) the affinity of the receptor for the hormone (how much hormone do you need to get receptor binding?)

27. Regulation of Receptor Number: the Phenomenon of Spare Receptors We know that cells typically have about 20 times more receptors than is needed for a maximal biological response. A complete biological response occurs after binding to only 5% of the receptors on a cell. This remaining 95% are called “spare receptors”. Why have spare receptors? Biological Response (% max) Receptor Occupancy (%) 100 50 0 0255075100

28. Effect of Decreasing Receptor Number in a Cell Which Does Not Have Spare Receptors No change in affinity. Decrease in maximal biological response. Hormone Concentration (M) % of receptors occupied % maximal response 100 75 50 25 0 100 75 50 25 0 10 -11 10 -10 10 -9 Kd

29. Effect of Decreasing Receptor Number in a Cell Which Has Spare Receptors In this example, assume you need 5000 receptors occupied for maximal biological response. If you start w/ 20,000 receptors occupied, decreasing receptor number does not change receptor affinity (Kd). Number of receptors bound 20,000 15,000 10,000 5,000 0 50 75 % Reduction in Receptor Number Kd # Receptors for maximal biological response Hormone Concentration (M)

30. Effect of Decreasing Receptor Number in a Cell Which Has Spare Receptors No change in maximal biological response (unless you go below 5000 receptors/cell). Requires higher dose of hormone to obtain maximal response. Hormone Concentration (M) 10 -11 10 -10 10 -9 10 -8 10 -7 % Maximal Biological Response 100 80 60 40 20 0 20,000 R/cell 10,000 5000 2500

31. Hormones, Agonists, and Antagonists Substances other than a receptors normal hormone may exist (or be made). Each substance that binds to a receptor has an intrinsic activity (related to the resulting biological response). SubstanceIntrinsic Activity hormone100% (by definition) superagonist>100% partial agonist<100% antagonist0%

32. Antagonists If a substance binds to a receptor but does not cause a biological response, it blocks the natural hormone from binding to it. Example: RU486 binds to the progesterone receptor, but does not cause a response. hormone binding domain transcriptional domain PR RU486 progesterone DNA

33. Regulation of Biological Response at the Receptor Level The strength of signal transduction can be regulated at the level of the receptor by several mechanisms: 1) change the affinity of the receptor (make it bind more difficult or easier to bind hormone). This usually doesn’t happen. 2) change the numbers of receptors on the cell (common) - internalization and degradation of receptors - occupancy of receptors (prevents hormone binding) - gene expression/synthesis 3) change the signal transducing ability of the receptor (usually for rapid regulation) - phosphorylation (usually inhibits receptor activity) - G protein uncoupling (stay tuned)

34. Plasma Membrane-Bound Receptors Recall that peptide hormones are polar, and cannot readily cross the cell membrane. Therefore, their receptors must be on the outside surface of the cell.

35. Types of Plasma Membrane Receptors There are five basic types of membrane bound receptors (grouped by signal transduction method): tyrosine kinase receptors receptors that are closely linked to tyrosine kinases receptors with guanylyl cyclase activity receptors that serve as transporters G protein-coupled receptors

36. Signal Transduction by Plasma Membrane Receptors 1) Receptors with intrinsic tyrosine kinase activity. Binding of hormone to the receptor induces the phosphorylating activity of the receptor. Example: Insulin receptor plasma membrane extracellular domains (ligand binding) tyrosine phosphorylase domains phosphorylated enzyme (altered activity)

37. Signal Transduction by Plasma Membrane Receptors 2) Receptors that are closely linked to tyrosine kinases. These activate cytoplasmic tyrosine kinase enzymes. Example: Growth Hormone Receptor associated tyrosine kinase phosphorylated enzyme

38. Signal Transduction by Plasma Membrane Receptors 3) Receptors with Guanylyl Cyclase Activity. Binding to the receptor activates guanylate cyclase region of the receptor, causing conversion of GTP to cyclic GMP. Example: Atrial Natriuretic Peptide Receptor guanylate cyclase GTP cyclic GMPprotein kinase G ion channels phosphodiesterase levels

39. Signal Transduction by Plasma Membrane Receptors 4) Receptors that serve as transporters. These move the ligand inside the cell, where they have an effect. (Not typical for hormones). Example: Iron, transported by transferrin receptor iron transferrin

40. Signal Transduction by Plasma Membrane Receptors 5) G Protein-coupled receptors. (The largest group!) These receptors are coupled with guanine nucleotide- binding proteins (G proteins), which activate various signaling pathways. Examples: Receptors for LH, FSH, TSH, GnRH, dopamine, serotonin, glutamine, parathyroid hormone, interleukins, etc.

41. G Protein-Coupled Receptor Superfamily Common structural features: - an amino terminus hormone-binding domain - seven hydrophobic transmembrane domains - a carboxyl terminus, intracellular domain

42. G Protein-Coupled Receptor Superfamily Common functional features: - binding to the receptor activates a G protein - each receptor is associated with a specific type of G protein - each G protein type has different functions: - Gs: stimulates cyclic AMP - Gi: inhibits cyclic AMP - Go activates phospholipase C

43. How G Proteins Work G proteins are composed of three subunits: alpha (  ), beta (  ), and gamma (  ). In the inactive state, the three subunits are associated with the receptor at the plasma membrane. The alpha subunit has a guanosine diphosphate attached.    GDP NH2 COOH

44. How G Proteins Work (cont.) When hormone binds, the GDP leaves the alpha subunit, and is replaced by a GTP. The alpha subunit then goes off to activate signaling pathways.    GDP NH2 COOH hormone GTP    NH2 COOH hormone GTP signal pathways

45. How G Proteins Work (cont.) After activating the signal pathway, the GTP is hydrolyzed into GDP, and the alpha subunit returns to the beta and gamma subunits at the membrane. GTP  P    GDP NH2 COOH

46. G Protein Stimulation of Cyclic AMP Binding of many hormones to their receptors results in the stimulation of the second messenger, cyclic AMP. G Protein involved: Gs

47. G Protein Stimulation of Cyclic AMP GTP a b ATP cAMP a b GTP GTPase GDP AC LHR Gs    GTP GDP a b LH

48. Receptor-G protein Interactions How are receptor-G protein interactions measured? Ligand-binding assays: High-affinity Low- affinity RG (GDP) GDP GTPγS R + G (GTP-δ-S) Without GTP, both high- and low-affinity states are measured. With GTP and Mg 2+, only low-affinity state is measured, because Agonist binding rapidly induces change from high- to low-affinity.

49. How Else Is Cyclic AMP Regulated? In addition to regulating the production of cyclic AMP, there is also regulation of its degradation. Degradation of cyclic AMP is by phosphodiesterases, which break down cyclic AMP into 5’-AMP Inhibitors of phosphodiesterases prolong activation of the cyclic AMP system.

50. Regulation of cAMP Levels by Phosphodiesterases a b GTP ATP cAMP 5’-AMP PDEs PDE Inhibitor X (-)

51. Protein Kinase A Pathway Increased cyclic AMP activates protein kinase A. Protein kinase A (PKA) is composed of two regulatory subunits and two catalytic subunits. Binding of cyclic AMP to the regulatory subunits frees the catalytic subunits, which have kinase activity. PKA uses ATP to phosphorylate specific enzymes in the cell, influencing their activity. regulatory catalytic cyclic AMP

52. Effects of Cyclic AMP-dependent PKA on Gene Transcription Cyclic AMP regulates the transcription of many genes by increasing PKA and causing the phosphorylation of the Cyclic AMP Response Element Binding Protein (CREB). CREB is a transcription factor which binds to a consensus cyclic AMP-response element (CRE) on the 5’-flanking region of many genes. intron exon CRE ERE TATA BOX CAT 5’-flanking region

53. CREB The CRE has a palindromic consensus sequence: 5’-TGACGTCA-3’ 3’-ACTGCAGT-5’ Removing the CRE from cyclic AMP-responsive genes causes a loss of regulation by cyclic AMP. Adding a CRE to non-cyclic AMP-responsive genes confers responsiveness to cyclic AMP. CREB binds to the CRE as a dimer. Phosphorylation increases the dimerization of CREB, resulting in increased transcriptional activity. Cells deficient in PKA cannot transcribe genes via the CRE (phosphorylation of CREB is required)

54. Terminology: CRE(cyclic AMP response element); CREB: CRE binding protein; CBP: CREB binding protein

55. CREM More recently, Cyclic AMP-Response Element Modulators (CREMs) has been identified. Structurally related to CREB. Four isoforms exist, all the product of a single gene. Three isoforms block cyclic AMP-dependent gene transcription. One isoform is an agonist for the CRE. Relative expression of isoforms is regulated in the testis: - immature sperm cells express antagonist form - maturing sperm cells express agonist form

56. Summary of Cyclic-AMP Signaling hormone binds receptor CREB initiates transcription Gs  activates adenylyl cyclase adenylyl cyclase produces cyclic AMP cyclic AMP activates PKA PKA phosphorylates CREB cAMP-GEFs Ras/Rap PKB/SgK

57. Why Make it So Complicated? Many steps = many places where regulation can take place In addition, at several steps the signal is AMPLIFIED. For example, activation of adenylyl cyclase produces several cyclic AMP molecules. Each activated PKA can phosphorylate many CREB molecules.

58. Influence of G Proteins on Phospholipase C Receptors coupled to G o activate phospholipase C, which hydrolyzes an inositol phospholipid into inositol triphosphate (IP 3 ) and diacylglycerol (DAG). IP 3 and DAG each activate separate signaling pathways: IP 3 activates a Ca 2+ pathway. DAG activates the protein kinase C pathway.

59. Actions of IP 3 and Ca 2+ IP 3 causes release of intracellular Ca 2+ stores (ER) and allows extracellular Ca 2+ to enter the cell. Result: increased free cytoplasmic Ca 2+. Ca 2+ can then bind to calmodulin, activating it. Calmodulin influences the activity of other enzymes, including kinases. GoIP3Ca 2+ calmodulin

60. Gq signaling pathways, Ca 2+, IP 3, PKC

61. Actions of DAG and PKC DAG activates calcium-dependent Protein Kinase C (PKC), by increasing PKC’s affinity for calcium. PKC phosphorylates a number of enzymes at serine/threonine residues, influencing their activity. PKC activates the transcription factor AP-1: - jun/fos heterodimer - binds to AP-1 sites on the 5’-flanking region of genes (similar to CRE, but different!) - binding influences gene transcription

62. Summary of G o Signaling G o  IP3calciumcalmodulin DAG PKC AP-1 gene transcription enzyme activity

63. Next Lecture….. Intracellular Hormone Receptors