Cell communication

• principles of cell communication

  • Most signals are extracellular and must bind a receptor to be functional
  • Signals that can’t pass the membrane must transmit their signal by a change in the receptor’s shape (conformation)
  • The signal is amplified in the cell
• Signal transduction

  • The process by which information from extracellular molecules is translated to an internal cellular signal either by electrical or chemical (hormone, neurotransmitter, etc) means.
• Receptor based signaling

  • Target cells contain the receptor which binds specific signals (ligands)
  • Receptors are usually transmembrane
  • Receptors can be cytosolic - the ligand must cross the membrane on its own
• What are the 4 different kinds of signaling mechanisms?

  • Juxtacrine: ligand is membrane bound on one cell and the receptor is membrane bound on another cell. Both cells have to be in close proximity for them to touch and a signal to be passed.
  • Autocrine/paracrine: a cell produces a ligand and the signal then binds to a receptor on the same cell (autocrine) or a neighbouring cell (paracrine)
  • Endocrine: longest range signaling. Ligand is carried through the blood to target cells throughout the body. eg. estrogen, testosterone, and prolactin
• identifying classes of Receptors

  A) GCPR

  B) cell surface receptor

  C) Receptor tyrosine kinase

  D) steroid hormone receptor

  E) cytokine receptor

  F) ion channel

  
• 6 Classes of receptors

  • GCPR
  • cell surface
  • receptor tyrosine kinase
  • steroid hormone receptor
  • cytokine receptor
  • ion channel receptor
• Basic elements of cell signaling systems

  • Receptors on or in target cells receive an extracellular message.
  • Ligand – molecule that binds to the receptor
  • Second messengers produced by effector in response to signal that amplify the signal
  • Cytoplasmic protein recruitments, most often involving kinases that activate or inactive proteins through phosphorylation
• Signal transduction using second messengers

  Second messengers are generally non-protein molecules that amplify the signal in the cell.
• Principles of signal transduction using a cascade of protein kinases and phosphatases

  • Signaling pathways consist of a series of proteins. Each protein in a pathway alters the conformation of the next protein.
  • Protein conformation is usually altered by phosphorylation.
  • Kinases add phosphate groups while phosphatases remove them.
  • Target proteins ultimately receive a message to alter cell activity.
  • This overall process is called signal transduction.
• Kinases can phosphorylate multiple kinases which then go on to phosphorylate more kinases until it phosphorylates a transcription factor that will cause transcription of a gene
• Reversible phosphorylation

  protein kinases transfer a phosphoryl group from ATP to a substrate protein

  • Ser, Thr, Tyr, His or Arg

  Protein phosphatase removes it
• Control of signal transduction

  • Cell type specificity of ligand-specific receptors
  • Timing of docking protein activation
  • Presence/absence of docking sites
  • Inhibitory proteins prevent certain signals
• Prolactin-Jak-STAT (Y kinase) Pathway

  • There are two prolactin receptors each attached to a Jak2
  • Prolactin binds to the prolactin receptor (cytokine receptor) causing the two receptor chains undergo a conformation change that moves the chains closer together
  • When the chains get closer together, there is activation of Jak2 by phosphorylation
  • JAK2 phosphorylates the receptors which help fully activate the receptor and create docking sites
  • STAT5 bind to receptor and are phosphorylated by Jak2 which allow STAT5 to dimerize.
  • STAT5 dimers transduce signals and act as transcription factors
• How is the Jak2 signaling pathway turned off?

  phosphatases
• How do the phosphorylated STAT5 molecules interact with each other?

  SH2 domain allows proteins to bind to phosphorylated tyrosines (on STAT5)
• How does STAT5 know when prolactin is bound?

  Prolactin induces a conformation change in the receptors which activates the Jak2s, the Jak2s then phosphorylate each other (autophosphorylation) and the receptors. Jak2 then phosphorylate the STAT5's.
• What is the active and inactive form of G proteins and how does the G-protein move between the two states?

  Active: GTP bound

  Inactive: GDP bound

  Active to inactive: GTP hydrolysis

  Inactive to active: release of GDP and binding of GTP (nucleotide exchange)
• G proteins are common switches to regulate signal events. Based on second messenger based signalling
• How do GPCR work together with heterotrimer G-proteins to relay signals?

  Heterotrimeric G-proteins have three different polypeptide subunits (α, β, ϒ)

  • they relay signals from ligand-bound receptors (active) to the cytoplasm/nucleus via an effector protein
  • α and ϒ are anchored into the membrane by lipid groups
• How do G proteins activate effectors in the example of adenyl cyclase?

  1. the ligand binds to the receptor, altering conformation and increasing its affinity for the G proteins
  2. GTP binding site is on the α subunit
  3. 2. Gα releases its GDP and as GTP binds. The nucleotide exchange results in a conformational change in the Gα subunit (less affinity for Gβϒ). Gα attaches to effector to activate the effector
  4. 4. Effector produces second messengers (eg. cAMP) that activate one or more signaling proteins (cascade effect).
• How is the activation response of effectors by GCPRs terminated?

  1. GTP on Gα is hydrolyzed causing a conformational change which allows the Gα to dissociate from the effector and reassociate with the Gβϒ dimer to form an inactive heterotrimer G protein.
  2. Receptor still active and active conformation makes it easy for G protein coupled receptor kinase (GRK) to phosphorylate it.
  3. The phosphorylated receptor makes for a good docking site for arrestin which prevents receptor from activating more G proteins.
  4. receptor endocytosed and 2nd messengers degraded by arrestin
• cAMP as a 2nd messenger

  Second messengers allow a wider response from a single extracellular first messenger

  • In the case of glucose mobilization, heterotrimeric G-proteins activate adenylylcyclase (an effector) that catalyzes cAMP from ATP.
  • cAMP is broken down by phosphodiesterase to AMP
• What 3 hormones (ligands) activate adenyl cyclase?

  • adrenocorticotropic hormone (ACTH) which controls cortisol production (liver)
  • glucagon which increase blood glucose levels (liver)
  • epinephrine which triggers flight or fight (skeletal and cardiac muscle)

  adenyl cyclase has 3 ligands and 3 receptors
• First steps to the response of a liver cell to glucagon or epinephrine


  • The reaction cascade occurs as the hormone binds to its GPCR
  • Ga subunit activates adenylyl cyclase which forms of cAMP molecules
  • Diffusion into the cytoplasm where they bind a cAMP-dependent protein kinase, protein kinase A (PKA)
  • PKA inhibits glycogen synthase so less glycogen is made, turns on genes that produce glucose, and activates pathways that break glycogen down into glucose
• Points of amplification as the liver cell binds to glucagon or epinephrine


  Binding of a single hormone molecule can activate a number of G proteins, each of which can activate an adenylyl cyclase effector, each of which can produce a large number of cAMP messengers quickly.

  • The production of a second messenger provides a mechanism to greatly amplify the signal generated from the original message.
• How does PKA effect the nuclear aspects of glucagon/epinephrine in liver cells

  PKA can translocate into the nucleus to phosphorylate key a transcription factor called cAMP response element-binding

  protein, (CREB).

  • Phosphorylated CREB binds as a dimer to CRE elements (cAMP response element) on DNA
  • a pathway by which glucose is formed from the intermediates of glycolysis, are encoded by genes that contain nearby CREs.
• How are some 2nd messengers generated from lipids?

  Phosphatidyl-inositol can be converted to other phosphorylated derivatives by the PH domain (pleckstrin homology domain) of phospholipase-C which binds to the phosphorylated inositol ring of a phosphoinositide.

  • phosphatidylinositol-specific phospholipase-C (Pi-PLC) is activated by acetylcholine GCPRs on smooth muscle who's G proteins activate the effector, Pi-PLC.
  • Pi-PLC cuts the phosphatidylinositol 4,5 P2 in half to form:
  • IP3 (inositol 1,4,5-triphosphate) and diacylglycerol (DAG) which act as 2nd messengers
• diacylglycerol (DAG)


  DAG, a plasma membrane lipid molecule, recruits and activates effector proteins that bear a DAG-binding C1 domain like protein kinase-C (helps with smooth muscle contraction)
• Inositol 1,4,5-Triphosphate (IP3)


  IP3 formed at the membrane diffuse into the cytosol and bind to a specific IP 3 receptor located at the SER.

  • The IP 3 receptor is a tetrameric Ca 2+ channel. Binding of IP 3 opens the channel, allowing Ca 2+ ions to diffuse into the cytoplasm.
  • Ca2+ ions are 2nd messengers.
  • Smooth muscle cell contraction is triggered by elevated Ca2+ levels
• How can Ca2+ levels be visualized in a living cell?

  • Fluorescent calcium binding compounds (eg. fura2)
  • Calcium-sensitive, light-emitting molecules.
• The Role of Calcium as an Intracellular Messenger

  • Unlike cAMP, Ca2+ can activate a number of effectors via calcium-binding proteins
  • The best-studied calcium-binding protein is calmodulin, which contains four binding sites for calcium.
  • If Ca2+ concentration rises, the ions bind to calmodulin, changing the conformation of the protein and increasing its affinity for a variety of effectors.
  • It can activate binding proteins such as kinases, phosphodiesterase (can turn off cAMP pathway), ion channels or calcium transport mechanisms
• types of tyrosine kinase receptors and receptor activation?

  Types of receptors:

  • receptor tyrosine kinases (RTKs); are directly activated by extracellular signals and has a ligand binding domain
  • cytoplasmic protein-tyrosine kinases: regulated indirectly by the ligand (eg. Jak2)

  Types of receptor activation:

  • ligand mediated activation
  • receptor mediated activation
• Steps in the activation of RTK

  Two mechanisms for receptor dimerization:

  • ligand-mediated dimerization (e.g., PDGF platelet derived growth factor), which has one ligand with 2 receptor-binding sites.
  • This brings receptors closer together and activate them
  • receptor-mediated dimerization (e.g., EGF epithelial GF). Each receptor chain binds one ligand.
  • This brings receptors closer together and activate them

  For most RTKs, dimerization brings two kinase domains in close contact for trans-autophosphorylation and other areas of the receptor chain to create docking sites
• Protein kinase function and autophosphorylation


  • Kinase activity is usually controlled by autophosphorylation on tyrosine residues that are present in the activation loop of the kinase domain.
  • Following its phosphorylation, the activation loop is stabilized in a position away from the ligand-binding site, resulting in activation of the kinase domain.
  • The receptor subunits then phosphorylate each other on tyrosine residues that are present in regions adjacent to the kinase domain; these sites act as binding sites for cellular signaling proteins.
• Protein-Protein interactions with docking sites and partners

  • SH2 domain - Src homology domain 2 – binds phosphorylated tyrosines
  • SH3 domain - Src homology domain 3 – binds hydrophobic proline residues
  • PTB domain - phosphotyrosine-binding domain – binds phosphorylated tyrosines
• Signaling proteins of RTKs

  • Adaptor proteins – GRB2 - binds to receptor to recruit other proteins to adaptor protein. Has both SH2 (bind to RTK receptor) and SH3 (binds to other proteins)
  • Docking proteins – IRS (insulin receptor substrate) creates 4 extra dockings sites on receptor
  • Transcription factors – STATs
  • Signaling enzymes - PLC (2nd messengers), lipid kinases, phosphatases, GTPase activating proteins
• Accessory proteins that modulate the activity of small G-proteins:

  • Guanine nucleotide-exchange factors (GEFs)- stimulate dissociation of the bound GDP, promoting GTP binding and activation of G-proteins
  • GTPase-activating proteins (GAPs)- stimulate hydrolysis of the bound GTP by the G-protein, decreasing the duration of the signal and help inactivate the G protein
  • guanine nucleotide-dissociation inhibitors (GDIs) – inhibit the release of bound GDP, maintaining the inactive state of G protein
• GTPase-activating proteins shortens the active time of RAS
• RAS is a small monomeric G protein


  • SOS helps recruit RAS
  • GAPs shorten the active time frame of RAS
• Docking proteins such as IRS provide extra versatility, how?

  Docking proteins such as IRS provide additional phosphorylation sites and extra versatility.

  • IRS contains a PTB (or others an SH2) to bind the receptor and is then phosphorylated by the receptor to provide extra docking sites