Part I => CARBS and LIPIDS. 1.7 Signal Transduction 1.7a Endocrine Hormones 1.7b Hormone Receptors

Transcription

1 Part I => CARBS and LIPIDS 1.7 Signal Transduction 1.7a Endocrine Hormones 1.7b Hormone Receptors

2 Section 1.7a: Endocrine Hormones

3 Synopsis 1.7a - Hormones are chemical messengers that play a key role in cellular signaling they differ from growth factors and cytokines in that they are exclusively produced by endocrine glands and travel in the blood to act at distant sites (systemic action) - On the other hand, growth factors and cytokines are produced throughout the body by numerous cell types and they can act both locally and systemically - Be aware that the terms hormones, growth factors and cytokines are often used interchangeably as there is no rigid boundary separating their classification and their overlapping functions only add to the confusion - The effect of endocrine hormones (as well as growth factors and cytokines) is almost always mediated via a cascade of signaling proteins and receptors, one or more of which are usually enzymes - While there is a diverse array of endocrine hormones, they can be broadly divided into two major categories on the basis of their chemical structure: (1) Micromolecular hormones these are small molecules such as the steroid hormones (2) Macromolecular hormones these are peptides and proteins such as insulin and glucagon

4 Endocrine System: Endocrine Glands - Glands are specialized organs in the body that synthesize and secrete chemical products such as sweat and insulin - Glands are divided into two major categories exocrine and endocrine - Exocrine glands secrete their products via ducts to specific internal or external sites examples are sweat glands and salivary glands (also mammary glands) - Endocrine (ductless) glands secrete their products directly into the bloodstream examples include pituitary gland, thyroid gland, and adrenal glands (also ovaries and testes) - Not all glands are monolithic eg pancreas acts both as an endocrine and exocrine gland - The islets of Langerhans (2%) of pancreas act as an endocrine gland and secrete metabolic hormones such as insulin and glucagon directly into the bloodstream - However, the bulk (98%) of pancreas is an exocrine gland that secretes via the pancreatic duct a cocktail of digestive enzymes such as trypsin and chymotrypsin into the small intestine

5 Endocrine System: Endocrine Signaling - In response to specific stimuli, the ductless endocrine glands secrete hormones directly into the bloodstream so that they can be carried to a distant site (target cells and tissues) - Upon arrival at their target site, endocrine hormones elicit specific biological effects such as the maintenance of homeostasis (homeo same ; stasis static/steady ) a steady-state in which living cells tend to maintain a relatively stable and constant environment via regulation of factors such as temperature (thermoregulation), glucose (glucoregulation), salt and water balance (osmoregulation)

6 Endocrine System: Micromolecular Hormones Cortisol (Adrenal glands) Aldosterone (Adrenal glands) Testosterone (Testes) Estradiol (Ovaries) Class Example Receptor Major stimuli Principal Function Progesterone (Ovaries) - Micromolecular hormones such as steroid hormones regulate numerous physiological functions central to homeostasis - Steroid hormones (highly hydrophobic/lipophilic) exert their effects by virtue of their ability to diffuse through the membrane and binding to their specific intracellular (cytoplasmic and nuclear) receptors called steroid hormone receptors (SHRs) - SHRs are a subfamily of nuclear receptor superfamily a group of transcription factors that become activated upon the binding of a ligand such as a hormone or a vitamin - Steroid hormones are subdivided into five major classes according to their physiological functions: Glucocorticoids Cortisol Glucocorticoid receptor Stress, hypoglycemia Metabolism and inflammation Mineralocorticoids Aldosterone Mineralocorticoid receptor Hypotension, acidosis Osmoregulation salt and water balance Androgens* Testosterone Androgen receptor Exercise, being stress-free Male sex steroid Estrogens* Estradiol Estrogen receptor Exercise, being stress-free Female sex steroid Progesterones* Progesterone Progesterone receptor Exercise, being stress-free Menstruation, pregnancy & embryogenesis *Produced in both males and females but in reciprocal quantities!

7 Endocrine System: Macromolecular Hormones (29aa) Glucagon (PDBID 1GCN) - Macromolecular hormones such as insulin and glucagon secreted by the islets of Langerhans of pancreas regulate blood sugar level (glucoregulation) -chain (30aa) S-S -chain (21aa) - Secreted in response to high blood glucose level, insulin aids the absorption of glucose from the bloodstream by muscle and adipose cells as well as inhibiting the production of glucose by the liver insulin exerts such effect by virtue of its ability to bind and activate the cell surface insulin receptor a member of the receptor tyrosine kinase (RTK) family S-S Insulin (PDBID 4NIB) - Secreted in response to low blood glucose level, glucagon does the opposite of insulin it stimulates the liver to release glucose through the breakdown of glycogen (glycogenolysis) and the synthesis of glucose from non-carbohydrate precursors such as pyruvate and lactate (gluconeogenesis) glucagon exerts such effect by virtue of its ability to bind and activate the cell surface glucagon receptor a member of the G-protein-coupled receptor (GPCR) family - Upon their activation, insulin and glucagon receptors set off a cascade of downstream events within the cytoplasm to transduce the insulin or glucagon message into the desired response - From a homeostatic perspective, insulin and glucagon act antagonistically to maintain a relatively constant blood glucose level (~1mg/ml) irrespective of external food intake in non-diabetics

8 Exercise 1.7a - Explain why only certain cells respond to hormones even though all cells in the body are exposed to the hormone - List hormones produced by the pancreas and adrenal glands. What types of molecules are these hormones? - Summarize the biological effects of insulin and glucagon - Summarize the biological effects of steroid hormones

9 Section 1.7b: Hormone Receptors

10 Synopsis 1.7b - How do endocrine hormones drive cellular processes as diverse as cell growth and cell proliferation through metabolism to the development and progression of many cancers? Enter hormone receptors! - Hormone receptors mediate extracellular signals in the form of hormones at the cell surface (or after their diffusion into the cytosol) to downstream targets such as transcription factors in the nucleus via what are termed signaling cascades - Hormone receptors and many proteins involved in coupling them to downstream cellular targets are modular ie they are functionally subdivided into semi-autonomous regions called modules or domains - Among a wide plethora of protein modules, Src homology domains such as SH2 and SH3 play a central role in directly coupling activated hormone receptors to downstream targets - Hormone receptors can be broadly divided into three major categories: (1) Steroid hormone receptors (SHRs) (2) Receptor tyrosine kinases (RTKs) (3) G-Protein-coupled receptors (GPCRs)

11 (1) SHRs: Structural Organization N TA DB LB C - Steroid hormone receptors (SHRs) a subfamily of nuclear receptor superfamily (~50 members in humans) are water-soluble intracellular (cytoplasmic and nuclear) proteins that act as ligand-modulated transcription factors ie they require a hormone for activation - SHRs are generally comprised of the TA-DB-LB modular architecture (though alternative but highly illogical nomenclatures also float around in the literature): DNA-Binding (DB) domain Binds to promoters of target genes in a sequence-dependent manner but only upon ligand binding to the LB domain! Ligand-Binding (LB) domain Recruits other cellular proteins such as transcription factors, co-activators and co-repressors to gene promoters in a ligand-dependent manner hormone binding to the LB domain is a pre-requisite for such functional output Transactivation (TA) domain Synergizes the action of LB domain by recruiting additional cellular proteins required for the assembly of fully functional transcriptional machinery at the target gene promoters unlike the LB domain, the TA domain acts in a ligand-independent manner but its role is essential

12 (1) SHRs: A Typical Signaling cascade - SHRs usually exist as monomers in complex with heat shock proteins (HSPs) in the cytoplasm Steroid Hormone - After diffusion through the cell membrane, the binding of the hormone to the LB domain results in its dimerization LB DB + LB DB LB LB SHR DB DB - Dimeric SHR translocates to the nucleus and binds to the target gene promoters via its DB domain TA HSPs TA Nucleus TA TA - Recruitment of cellular factors required to assemble the transcriptional machinery at the gene promoters is aided by the LB and TA domains - This turns on gene expression of specific proteins which in turn set about causing changes to the cell in response to the hormone DNA TA LB LB DB DB TA Transcriptional machinery mrna

13 (2) RTKs: Structural Organization Extracellular LB LB LB LB Lipid Bilayer TM TM ATP Ligand Binding ADP TM TM Cytoplasmic TK TK Dimerization & Autophosphorylation P TK TK P - Receptor tyrosine kinases (RTKs) are single-transmembrane cell surface receptors comprised of: (1) Extracellular ligand binding (LB) domain (2) Single -helical transmembrane (TM) domain (3) Cytoplasmic tyrosine kinase (TK) domain - Upon the binding of a cognate ligand (such as a hormone, cytokine or growth factor) to extracellular LB domain, the receptors either dimerize or undergo a conformational change so as to bring their cytoplasmic TK domains close together such proximity and orientation allows each TK domain to phosphorylate its dimeric a specific Tyr residue in a trans-fashion ie each TK phosphorylates the other and vice versa! - Autophosphorylation of RTKs in such a manner results in their activation, thereby allowing them to recruit specific signaling proteins to the site of inner membrane surface this in turn sets off a cascade of downstream events ultimately culminating in the nucleus

14 (2) RTKs: Autophosphorylation ATP ADP Kinase Phosphatase - Protein one of the hydroxy amino acids (eg Tyr, Thr or Ser) within protein chains is by far the most ubiquitous form of post-translational modification (PTM) observed in proteins why so? - Protein phosphorylation essentially serves as a molecular switch in that it can turn protein function ON or OFF courtesy of protein kinases and phosphatases working in tandem to maintain cellular homeostasis - Such molecular switches are a hallmark of the living machinery, and particularly, the cellular signaling pathways that need to be tightly regulated in a highly spatial and temporal manner ie with respect to both the location of the signaling event (spatial) and the short time that it should be turned on or off (temporal/transient) for the failure to do so often forms the basis of the development and progression of disease - The fact that the regulation of RTKs is also under the control of phosphorylation offers a fitting tribute to the importance of kinases and phosphatases to the vitality of life at molecular level the human genome encodes 500 protein kinases and 200 protein phosphatases!

15 (2) RTKs: Family Members - RTKs are one of the two subfamilies of the larger protein tyrosine kinase (PTK) superfamily comprised of close to 100 unique members the other being the nonreceptor tyrosine kinases (nrtks), or simply the cytoplasmic tyrosine kinases - Prominent members of the RTK family include: RTK Synonyms Protein/Peptide Ligand(s) Principal Function IR Insulin (I), insulin-like growth factors (IGFs) Glucose homeostasis EGFR ErbB1/HER1 Epidermal growth factor (EGF) Cell growth and proliferation PDGFR Platelet-derived growth factor (PDGF) Cell growth and differentiation FGFR Fibroblast growth factor (FGF) Cell growth and development VEGFR Vascular endothelial growth factor (VEGF) Development of blood vessels HGFR Met Hepatocyte growth factor (HGF) Embryonic development NGFR TrkA Nerve growth factor (NGF) Neuronal development EphR Ephs Ephrin (Eph) Cell adhesion and migration

16 (2) RTKs: Insulin receptor - Insulin receptor (IR) involved in the regulation of glucose homeostasis binds insulin and insulin-like growth factors such as IGF1 and IGF2 - IR is unusual in that it exists in a disulfide-linked dimeric state even in the unbound conformation ( and chains are covalently linked together via disulfide bridges) ligand binding merely causes a conformational change that promotes its autophosphorylation and subsequent activation

17 (2) RTKs: A Typical Signaling Cascade - Ligand binding to RTK induces receptor dimerization and/or autophosphorylation Ligand - Activated RTK serves as a binding site for the recruitment of adaptors such as GRB2 (via its P SH2 domain) to the inner membrane surface P (IMS) in a phosphorylation(tyr)-dependent manner - Since GRB2 adaptor exists in complex with SOS exchange factor, the recruitment of the guanine nucleotide exchange factor SOS to the IMS catalyzes GDP-GTP exchange in Ras, thereby resulting in its activation - Next, activated Ras binds and activates Raf kinase - Raf kinase then activates the kinase MEK via Ser/Thr phosphorylation RTK P P Extracellular SH2 GRB2 Jun SH3 SH3 SOS Ras GDP Cytoplasm Nucleus MAPK Ras GTP MAPK P MEK P Raf P mrna - This is followed by the activation of the MAP kinases (MAPKs) such as ERK2 by MEK, also via Ser/Thr phosphorylation P Jun - Activated MAPK translocates to the nucleus and phosphorylates specific transcription factors (eg Jun/Fos/Myc) - Phosphorylated Jun binds to its promoter within the target genes and turns on gene expression of specific proteins which in turn set about causing changes to the cell in response to the ligand

18 (3) GPCRs: Structural Organization - G-protein-coupled receptors (GPCRs) are seventransmembrane cell surface receptors with close to 1000 members they not only transduce hormone signals but also ligands/stimuli as diverse as light (eg rhodopsin), odors, pheromones and neurotransmitters - In the absence of a ligand, the cytoplasmic tail of GPCRs binds to the GDP-bound -subunit of the so called G-proteins which are membraneanchored heterotrimers of, and subunits such union between GPCRs and G-proteins locks the latter in an inactive state Ligand (L) L GPCR Inactive G-protein ( ) - Upon ligand binding (eg glucagon) at their extracellular face, GPCRs undergo a conformational change that results in GDP-GTP exchange within the -subunit, thereby allowing it to dissociate off both the GPCR cytoplasmic tail and its heterotrimeric partners and subunits GDP-GTP exchange - Such dissociation enables both the GTP-bound - subunit (as well as the / -heterodimer) acts as a modulator of other membrane-bound proteins such as adenylate cyclase (AC) and phospholipase C (PLC) Active

19 (3) GPCRs: Adenylate Cyclase (AC) Adenosine triphosphate (ATP) 3-5 -Cyclic adenosine monophosphate (camp) - Upon activation by the GTP-bound -subunit of G-proteins, the integral membrane protein AC catalyzes the conversion of ATP to camp - camp then acts as an intracellular secondary messenger in the cytosol in response to extracellular hormone (primary messenger) signal acting through the GPCRs - Within the cytosol, camp targets and modulates the activities of other cellular proteins such as protein kinase A (PKA) a kinase involved in the regulation of carbohydrate and lipid metabolism by virtue of its ability to phosphorylate Ser/Thr residues in target proteins so as to effect gene expression

20 (3) GPCRs: Phospholipase C (PLC) - Upon activation by the GTP-bound -subunit of G-proteins, the cytoplasmic peripheral membrane protein PLC catalyzes the hydrolysis of PIP2 (a membrane its glycero-phosphoester bond into IP3 and DAG - IP3 and DAG then act as intracellular secondary messengers in the cytosol in response to extracellular hormone (primary messenger) signal acting through the GPCRs - Within the cytosol, IP3 triggers the opening of Ca 2+ channels in the endoplasmic reticulum the sudden increase in cytosolic concentration of Ca 2+ together with DAG leads to activation of proteins such as protein kinase C (PKC) a kinase involved in modulating numerous signaling cascades by virtue of its ability to phosphorylate Ser/Thr residues in target proteins so as to effect gene expression

21 Exercise 1.7b - How does a receptor tyrosine kinase phosphorylate itself? - Summarize various members of RTK family, their ligands and functions - Summarize the roles of proteins such as Grb2, SOS, Ras, and various protein kinases involved in coupling activated RTKs to downstream cellular targets such as transcription factors in the nucleus - Explain why cells contain an array of protein phosphatases as well as protein kinases - Summarize the steps of signal transduction from a GPCR to phosphorylation of target proteins by PKA - What is the function of a secondary messenger such as camp?

22 Bottled Water Exposed a Convenience or Health Risk! Polyethylene Terephthalate (PET) - Plastic water bottles are typically made from a polymer called polyethylene terephthalate (PET) in combination with a myriad of other chemicals and metals such as antimony (Sb) - During storage, transportation and exposure to heat, these nasty chemicals directly (or after decomposition) leach into the water as carcinogens and can often act as endocrine disruptors - Endocrine disruptors mimic the action of endocrine hormones such as estrogen, thereby disrupting signaling pathways, promoting cancer and other cellular defects eg several lines of evidence suggest that sustained exposure to antimony (a metalloestrogen) contributes to the development of breast cancer - Not only tap water is FREE but it is better of the two evils but avoid water filters as they also contain nasty carcinogenic compounds (anything that is made from plastic is malastic to health!)