Mechanism of hormone action

Hormones Three types Proteins Lipids Amino acid derivatives Glycoproteins Small pepstides Large proteins Lipids Cholesterol derivatives Eicosanoids Amino acid derivatives

Hormones Receptors Innate by themselves Require mediation Binding sites for a hormone Very specific

Hormone receptors Two types Transmembrane Intracellular/nuclear Proteins regardless of the type Interaction between a hormone and a receptor Initial step of hormone action

Transmembrane receptors Protein hormones Unable to pass through the plasma membrane Size Charges Receptors must be located on the plasma membrane Extracellular domain for interaction with hormone Intracellular signaling system

Types of transmembrane receptors Receptors with multiple transmembrane domains Seven trans-membrane domain receptor No intrinsic enzymatic activity (C-terminus) Associated with intracellular proteins involved in signaling G-proteins Modification of extracellular domain (hormone binding site, N-terminus) Glycosylation Crucial for hormone binding

Trans-membrane domains (7) Alpha-helix Hydrophobic amino acids Loops Connect alpha helices May be linked by disulfide bridges (extracellular loop 1 and 2)

Intracellular/cytoplasmic domain Palmitoylation of some cysteine residues Attachment of fatty acids Fourth loop Site for phosphorylation

General structure of seven trans-membrane receptor Variations Amino acid sequences Variable length of N-terminus Affects binding of ligand/hormone

Intracellular signaling Generated when a hormone interacts with extracellular domain of the receptor Conformational change within the trans-membrane helices Exchange of GDP to GTP on the alpha-subunit of G-protein complex Activation of Ga subunit Dissociation of activated Ga from G-protein complex (bg)

Second messengers Cyclic nucleotides (cAMP and cGMP) cAMP Widely used secondary messenger Generated by adenyl cyclase Activated by activated Ga subunit of G-protein complex Activation of cyclic nucleotide-dependent protein kinases Protein kinase A (cAMP)

Secondary messengers Amplification of hormonal signals Binding of hormone to the receptor Activation of adenyl cyclase by activated Ga Activation of protein kinase A by cAMP Rapid clearance and inactivation Phosphodiesterases Inhibited by methylxanthines (caffeine, theophylline, and theobromine) Phosphoprotein phosphatases

How do we know that cAMP is a secondary messenger? Changes in production of cAMP after hormonal treatment Correlation between amount of cAMP being produced and cellular response to the hormone Inhibition of phosphodiesterase activity Presence of ligand but no effects Treatment with cAMP analogues/agonists Similar response to that of hormone

Types of G-protein complex Ga subunit (20 different types) Gs (stimulatory Ga) Gi (inhibitory Ga) Go (associated with orphan receptors in neurons) Gt (transducin found in retina, activates cGMP-specific phosphodiesterases) bg complex 4 or more

Identification of specific G-protein complex associated with particular receptor Structurally similar to each other Use of pertusis toxin (bacterial toxin) Uncoupling of G-protein complex from the receptor Gi is very susceptible

G-protein complex coupled with secondary messenger system other than cyclic nucleotides Generated through phospholipid metabolism Inositol triphosphate (IP3) Diacylglycerol (DAG) Arachidonic acid Activation of phospholipase C (PLC) by activated Ga

IP3 DAG Water-soluble Binds to protein kinase C Stimulate release of Ca DAG Binds to protein kinase C Activated by elevated Ca

Medical importance 65 % of prescription drugs target G-protein coupled receptors Variety of ligands

Other protein hormone receptors Transmembrane receptors with intrinsic tyrosine kinase activity Receptor tyrosine kinase Receptors for insulin and many growth factors Transmembrane receptors with associated tyrosine kinases Cytokine receptors Receptors for growth hormone and prolactin No intrinsic kinase activity Interaction between receptor and hormone causes recruitment and activation of tyrosine kinases associated with receptor

Receptor tyrosine kinase Approximately 100 receptor tyrosine kinases in human Highly conserved Domains Extracellular Hormone binding site Transmembrane Intracellular/cytoplasmic Tyrosine kinase activity

16 subfamilies Based on extracellular domain Variation on extracellular domain Interaction with variety of factors EGF, PDGF, and insulin

Activation of receptor Dimerization Dimeric ligand (two subunits) Each subunit binds to a receptor Two binding sites within a hormone One hormone interacts with two receptors

Activation of receptor Pre-existence as a dimer Receptor is a dimer Activated through interaction with ligand

Activation of receptor Conformational changes in the kinase domain Accessible to the substrate Autophosphorylation of tyrosine residues (3 in insulin receptor) Activation loop Triggers conformational changes ATP binding Interaction with intracellular proteins Phosphorylation of other proteins