Peptide/Protein Hormones

• Scheme for discussing hormone action

  1. Anatomical description of hormonal cell/gland
  2. chemical structure and biosynthesis of the hormone
  3. mode of hormone transport to target
  4. hormone interaction with target
  5. biological response to hormone
  6. physiological effects of the hormone system
  7. feedback mechanisms that regulate the hormone system
• Peptide/protein hormones - general informaiton

  • water soluble
  • possibly thousands
  • products of translation
  • variability in the number of post translational modifications
  • come in a wide variety of sizes
  • 3 amino acids -> multi subunit glycoproteins
• Peptide vs Protein

  Proteins and peptides are chains of amino acids. Primary difference is in size (proteins are larger).
• Peptid/protein hormones

  • Thyrotrophin releasing hormone (TRH)
  • Corticotrophin releasing hormone (CRH)
  • Arginine vasopressin (AVP)
  • Gonadotrophin releasing hormone (GnRH)
  • Prolactin releasing factor (PRF)
  • Oxytocin
  • Thyroid-stimulating hormone (TSH)
  • Adrenocorticotrophic hormone (ACTH)
  • Luteinizing hormone (LH)
  • Follicle-stimulating hormone (FSH)
  • Somatotrophin/growth hormone (GH)
  • Prolactin (PRL)
  • Melanocyte-stimulating hormone (MSH)
  • Insulin
• Site of production

  • produced in a variety of places
  • at the level of the cell, peptide and protein hormones are produced in the endoplasmic reticulum -> golgi apparatus for packaging
  • secreted from secretory granules
• biosynthesis

  • encoded by a specific gene which is transcribed into mRNA and translated into a protein precursor called preprohormone
  • may involve multiple genes
  • alpha and beta subunits of the glycoprotein hormones (TSH, LH, and FSH) are derived from different genes
  • (pre)prohormones are often post-translationally modified in the endoplasmic reticulum to contain carbohydrates (glycosylation)
  • folding
  • stability
• preprohormones contain signal peptides (hydrophobic amino acids) which guides them into the lumen of the rough ER. Once targeted, the signal sequence is removed to form prohormone. Prohormone is processed into active hormone and packaged into secretory vesicles.
• transcription of the DNA sequence into RNA -> excision of sequences (introns) from the initial DNA transcript and modification of the 3' and 5' terminals -> translation of the mRNA into protein -> the prohormone is cleaved into fragments, a process that normally occurs prior to secretion
• signal sequence


  the first amino acids that are translated from the mRNA template form a signal sequence.

  signal sequence + signal recognition particle -> rough ER -> protein synthesis continues into rough ER
• the initial amino acid sequence to be translated is the signal sequence that finds a signal recognition particle on the membrane of the rough ER
• • translation of the mRNA into a protein
  • starts in the cytosol with ribosome and other helpers
  • signal sequence is the first thing to undergo translation
  • signal sequence binds recognition particle on rough ER; translation is paused until it enters the lumen
  • signal sequence is also generally cleaved prior to the end of the process
  • therefore, a complete preprohormone rarely exists
  • the prohormone is cleaved into fragments, a process that normally occurs prior to secretion
• inside the ER, the protein moves into the Golgi apparatus by fission and fusion of the protein-containing vesicles in which the large prohormone is cleaved by peptidases into the bilogically active hormone and one or more fragments of the original molecule. fragments are frequently co-secreted with the active hormone.
• protein and peptide hormone synthesis requires:

  • transcription of gene(s)
  • post-transcriptional modification by excision of the introns
  • translation of the mRNA and post-translational modification of the original amino acid sequence

  as a result, more than one prohormone may be derived from a single gene. post-translational processing of a prohormone may result in the formation of different biologically active peptide fragments

  • usually tissue specific
• calcitonin

  • 32 amino acids produces in parafollicular cells of thyroid
  • reduces blood calcium
• calcitonin-gene related peptide

  • 37 amino acids
  • produced centrally and peripherally by neurons
  • DRG - transmission of pain
  • motor neuron - regeneration after injury?
  • implicated in migraines
• prohormones continued

  • may help ensure correct folding and formation of disulfide bonds
  • size of prohormone not related to size of other prohormones
  • size of prohormone not related to size of final hormone product(s)
• regulated secretion

  • stored and released in bursts when cell is stimulated
  • allows large amounts of hormone to be released in a short period of time
  • most common form of secretion for peptide and protein hormones
• constitutive secretion

  • hormone is not stored by cells
  • vesicles of hormone released as they are produced
• • sometimes prohormone is secreted
  • converted in the extracellular fluid into the active hormone
  • ex: angiotensin is secreted by liver and converted into active form by enzymes secreted by kidney and lung
• most peptide and protein hormones do not need chaperone molecules to circulate the body

  • ex: insulin-like growth factor - 1 (IGF-1): needs to join up with one of several binding proteins
• receptors

  • two key components
  • a ligand binding domain
  • non-covalently binds hormone
  • an effector domain
  • recognizes the presence of the hormone and initiates the biological response
  • second messengers may be generated/activated, which transfer the message to other players in the cell
• • proteins and peptides are water soluble, so they do not diffuse across the hydrophobic lipid cell membranes
  • parts of their receptors lie extracellularly and they couple with intracellular signal transducing molecules by traversing the cell membrane
  • the majority of classical protein and peptide hormone receptors are G-protein linked receptors
• g-protein subunits: g proteins that are associated with GPCRs are heterotrimeric, meaning they have three different subunits: an alpha subunit, a beta subunit, and a gamma subunit
• alpha subunit:

  • catalytic subunit
  • GTPase activity
  • binds either GTP (active) or GDP (inactive)
• beta and gamma subunits:

  • regulatory subunits
  • like to inhibit alpha
  • dissociate from alpha upon activation
• GPCRs

  • in the absence of a signal, GDP attaches to the alpha subunit
  • the entire G protein-GDP complex is believed to be associated with a nearby GPCR
  • extracellular hormone-receptor interactions induce dissociation of the associated G protein
  • causes a conformational change in the GPCR, which activates the G protein
  • GTP replaces GDP
  • beta/gamma and alpha dissociate
  • allows alpha to wiggle laterally through the membrane to interact with other things
• signal sequence: G-protein coupled receptors

  1. hormone binding
  2. GTP binding, alpha subunit release from beta/gamma subunits
  3. alpha subunit binding to effector enzyme
  4. generation of cyclic AMP (cAMP) or DAG/IP3
• dissociation of the g protein leads either to:

  • opening of ion channels in the membrane
  • activation of a membrane bound enzyme that stimulates (or inhibits) the production of a second messenger such as cyclic AMP or diacylglycerol and inositol triphosphate
  • these second messengers then activate serine/threonine kinases or phosphates
• example:

  • Peptide hormone + GPCR
  • activates protein kinase C (PKC) via the inositol pathway
  • releases the transcription factor, NF-kB from its inhibitory subunit through phosphorylation
  • NF-kB moves into the nucleus where, along with other transcription factors (TF), initiates transcription
• • not every protein/peptide hormone uses a GPCR
  • the second most common type of cell surface receptors is that used in the signaling of insulin, growth hormone, prolactin, most growth factors and cytokines
  • it is a transmembrane receptor with either:
  • inherent protein tyrosine kinase activity (on the intracellular domain)
  • associated with the intracellular molecules that have kinase activity
  • mostly serine/threonine kinase activity
• • binding of the hormone or growth factor to the extracellular domain results in receptor dimerization with an adjacent receptor
  • ligand binding initiates either autophosphorylation or phosphorylation of an associated enzyme
• • receptors that have inherent tyrosine kinase activity bind molecules that have a specific SH2 domain (src homology domain)
  • in turn, another accessory protein may be activated such as SOS (son of sevenless)
  • this can activate a small GTPase (monomeric g protein) known as Ras that essentially acts as a signal transduction switch
  • Ras activation can lead to phosphorylation of Raf, MEK and eventually to mitogen activated protein kinase (MAPK) which can initiate transcription (termed the MEK-MAPK pathway)
• • activation of a single g protein can affect the production of hundreds or even thousands of second messenger molecules
• • one especially common target of activated G proteins is adenylyl cyclase, a membrane associated enzyme that, when activated by the GTP bound alpha subunit, catalyzes synthesis of a second messenger cAMP from molecules of ATP
  • in humans, cAMP is involved in responses to sensory input, hormones, and nerve transmission among others
• adenylate cyclase (AC) activation -> generation of cAMP -> cAMP dependent activation of protein kinase A (PKA) -> cAMP binding to PKA regulatory subunits -> release of catalytic PKA subunits
• • phospholipase C is a common target of activated G proteins
  • this membrane associated enzyme catalyzes the synthesis of two second messengers - DAG and IP3 - from the membrane lipid phosphatidylinositol
• phospholipase C pathway (DAG)

  alpha subunit activation of phospholipase C (PLC) -> PIP2 conversion to IP3 and diacylglyceride (DAG) : DAG -> activate protein kinase C (PKC) -> regulation of protein
• phospholipase C pathway (IP3)

  IP3 -> calcium influx (via IP3 receptors in the ER and G-protein activity) Functions of calcium:

  1. PKC activation
  2. calmodulin activation
  3. regulates CaM kinases
  4. exocytosis
• G alpha s

  • stimulatory
  • activity on:
  • adenylate cyclase
• G alpha q

  • stimulatory
  • activity on:
  • PLC
  • Ca channel