Hormone Receptors on the Plasma Membrane Characteristics of Receptors in General Five Groups of Membrane-Bound Receptors The G Protein-Coupled Receptor

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  • Slide 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
  • Slide 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).
  • Slide 3
  • Specific Binding A receptor will only bind (recognize) a certain hormone, or closely related hormones. LH hCG FSH LH Receptors LHhCG FSH
  • Slide 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).
  • Slide 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
  • Slide 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
  • Slide 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.
  • Slide 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.
  • Slide 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.
  • Slide 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
  • Slide 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
  • Slide 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 -
  • Slide 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
  • Slide 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
  • Slide 15
  • Slide 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.
  • Slide 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 )
  • Slide 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
  • Slide 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?????
  • Slide 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.
  • Slide 21
  • Scatchard transformation Y-axis is Bound/Free (total radioligand-bound) X-axis Bound (pmol/mg protein) Straight lines are easier to interpret.
  • Slide 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.
  • Slide 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. De

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