Question

A) cAMP and CREB- how phosphorylation can regulate gene transcription?

B) Purpose of second messengers and what molecules are used as second messengers?

C) Describe G proteins structure, how are they activated, how they transduce the signal

D) Describe Tryosine Kinase Receptors structure, how they are activated, how they transduce the signal.

Answer 1

(A) CREB is also known as the cAMP-response element-binding protein which plays a key role as a cellular transcription factor. It binds to CRE (cAMP response elements) present in DNA and results in regulation of gene expression. CREB family members are believed to be important for learning and memory and contribute to neuronal adaptation to drugs of abuse and hormonal control of metabolic processes, including regulation of gluconeogenesis by hormones glucagon and insulin. For instance, in case of CREB1, it activates transcription of the target genes in response to a diverse array of stimuli, including peptide hormones, growth factors, and neuronal activity. CREB1 is critical for a variety of cellular processes, including proliferation, differentiation, and adaptive responses.

In case of CREB phosphorylation and induced signaling, a signal (stimulus) arrives at the cell surface, activates the corresponding G- protein coupled receptor, which leads to the production of a second messenger such as cAMP or Ca2+. This in turn activates a protein kinase which translocates to the cell nucleus, to activate a CREB protein. The activated CREB protein then binds to a CRE region, and is then bound to by CBP (CREB-binding protein) resulting in coactivation. This is the key mechanism by which CREB phosphorylation induced by cAMP results in regulating gene expression by allowing it to switch certain genes on or off.

(B) Secondary messenger are a critical part of cellular signaling process in which proteins of different kind are activated through generation of diffusible signaling molecules to participate in cellular processes. Second messengers are produced catalytically in response to the extracellular signals (primary messengers) and amplify their response, thus second messengers are a part of signal transduction cascades. They relay signals received at receptors on the cell surface — such as the arrival of protein hormones, growth factors, etc. — to target molecules in the cytosol and/or nucleus. They not only relay the signal but also serve to greatly amplify the strength of the signal. The three major classes of second messengers are:

• cyclic nucleotides (e.g., cAMP and cGMP)
• inositol trisphosphate (IP3) and diacylglycerol (DAG)
• calcium ions (Ca2+)

For instance, when cAMP acts as a secondary messenger in G-protein mediated signaling cascades, it results in changing the molecular activities in the cytosol, often using Protein Kinase A (PKA) — a cAMP-dependent protein kinase that phosphorylates target protein in order to turn on a new pattern of gene transcription. Another example is that calcium where is acts as the most common secondary messenger and triggers a variety of cellar events including apoptosis, muscle contraction, adhesion of cells to extracellular matrix, and many other events mediated by protein kinase C.

(C) G proteins are also known as guanine nucleotide-binding proteins They are a family of proteins that act as molecular switches involved in transmitting signals from a variety of stimuli outside a cell to its interior. Their activity is regulated by factors that control their ability to bind to and hydrolyze guanosine triphosphate (GTP) to guanosine diphosphate (GDP). When bound to GTP, they are 'on', and, when bound to GDP, they are 'off'.

The structure of G proteins includes a heterotrimeric molecule consisting of 3 subunits ?, ? and ?. They are bound to the cytoplasmic surface of the plasma membrane. They are activated by G protein-coupled receptors (GPCRs) that span the cell membrane. GPCRs are characterized by an extracellular N-terminus, followed by seven transmembrane (7-TM) ?-helices (TM-1 to TM-7) connected by three intracellular and three extracellular loops, and finally an intracellular C-terminus. The GPCR arranges itself into a tertiary structure resembling a barrel, with the seven transmembrane helices forming a cavity within the plasma membrane that serves a ligand-binding domain. The intracellular GPCR domain activates a particular G protein inside the cell upon receiving extracellular stimulus (signallling molecule binds on extracellular domain). Their classification as stimulatory or inhibitory is based on the identity of their distinct ? subunit. Furthermore, they are activated in response to a conformational change in the GPCR, exchanging GDP for GTP, and dissociating in order to activate other proteins in a particular signal transduction pathway.

The common mechanism of transduction involves:

• ? and ? subunits to remain associated as ? ? complex with the cytoplasmic surface of the membrane when the system is inactive or in resting state and GDP is bound to the ? subunit.

• Binding of a stimulus (GPCR activation) facilitates GTP binding to ? subunit and promotes dissociation of GDP from its place.

• Binding of GTP activates the ? subunit and ?-GTP is then thought to dissociate from ? and interact with a membrane bound effector.

• The process is terminated when the hydrolysis of GTP to GDP occurs through the GTpase activity of the ?-subunit.

• The resulting ?-GDP then dissociates from the effector, and reunites with ? ? completing the response cycle.

• Also, there are many classes of G? subunits: Gs? (G stimulatory), Gi? (G inhibitory), Go? (G other), Gq/11?, and G12/13 and so on. These behave differently (specific mechanism of action) in the recognition of the effector molecule (a similar mechanism of activation). The most common example is that of G?s. It activates the cAMP-dependent pathway by stimulating the production of cyclic AMP (cAMP) from ATP. This in turn activates PKA which further effects phosphorylation of downstream targets of signaling cascades.

(D) Tyrosine Kinase receptors are also known as RTKs which act as high-affinity cell-surface receptors for a number of cytokines, growth hormones and peptides. RTKs play an important role in the cell cycle, cell migration, cell metabolism and many other substantial cell functions.

RTKs exist as dimers or dimerize during binding to ligands. Receptor tyrosine kinases are single-pass, transmembrane proteins and bind extracellular polypeptide ligands (i.e. growth factors) and cytoplasmic effector and adaptor proteins to regulate biological processes. Ligand binding promotes receptor dimerization and autophosphorylation (activation of kinase activity) of receptor tyrosine residues. The resultant conformational change stabilizes the active kinase, and subsequent phosphorylation events form binding sites for downstream adaptor, scaffold, and effector proteins.

In the structure of the RTK: each monomer has a single hydrophobic transmembrane-spanning domain composed of 25 to 38 amino acids, an extracellular N terminal region, and an intracellular C terminal region.

The extracellular N terminal region exhibits a variety of conserved elements (immunoglobulin (Ig)-like or epidermal growth factor (EGF)-like domains, fibronectin type III repeats, or cysteine-rich regions) characteristic for each subfamily of RTKs. As these domains contain primarily a ligand-binding site, which binds extracellular ligands, the sites are specific for enabling binding to the respective extracellular ligand.

The intracellular C terminal region also shows the highest level of conservation and comprises of catalytic domains responsible for the kinase activity of the receptors. This enables them to catalyze autophosphorylation and tyrosine phosphorylation of substrates.

Transduction mechanism:

• When signaling molecules bind to RTKs, they cause neighboring RTKs to associate with each other, forming cross-linked dimers.
• Cross-linking activates the tyrosine kinase activity in these RTKs through phosphorylation — specifically, each RTK in the dimer phosphorylates multiple tyrosines on the other RTK (cross-phosphorylation or autophosphorylation).
• Once cross-phosphorylated, the cytoplasmic tails of RTKs serve as docking platforms for various intracellular proteins involved in signal transduction which have a particular domain — called SH2. This domain enables them to binds to the phosphorylated tyrosines in the cytoplasmic RTK receptor tails. More than one SH2-containing protein can bind at the same time to an activated RTK, allowing simultaneous activation of multiple intracellular signaling pathways.
• Ultimately, RTK activation brings about changes in gene transcription.

Since crosstalk occurs between intermediates and various signaling pathways in the cell, in case of RTK mediated signaling, the pathway becomes more complex as these signals travel from the membrane to the nucleus. Common signaling pathways activated downstream of RTK activation include RAF/MAP kinase cascades, AKT signaling and others.

Family of RTKs include epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), vascular endothelial growth factor receptor (VEGFR) and others.