Describe the structure of G-protein coupled receptors.
GPCRs contain 7 transmembrane domains, including 3 intracellular loops and 3 extracellular loops. The C terminal is intracellular and rich with hydroxy-containing amino acids. The terminal is important for receptor regulation and desensitization. There are around 800 GPCRs in the human genome, out of which half are olfactory receptors and poorly known. Idiosyncratic ADR is related to the activation of Mas-related G protein-coupled receptor X2 located on Mast cells.
The basis of G-protein signaling.
Ligand (agonist) binding to GPCR induces structural change, causing GDP-release from the alpha subunit of the G-protein heterotrimer and subsequent GTP-binding. GTP-binding is followed by the dissociation of the alpha subunit (GTP-bound) from the beta and gamma subunits. The alpha subunits then mediates effector activation.
Describe the G-alpha s/i signaling pathway.
Agonist binding to beta-2 adrenoreceptor activates the G-alpha s subunit. The subunit activates adenylyl cyclase, which catalyzes the conversion of ATP into cAMP. Increased cAMP levels activates PKA (cAMP-dependent protein kinase), which activates downstream enzymes, ion channels, and transcription factors. Increased cAMP levels also activates phosphodiesterase (PDE), which catalyzes the conversion from cAMP to 5’ AMP, deactivating it.
Agonist binding to the alpha-2 adrenoreceptor activates the G-alpha i subunit, which inhibits adenylyl cyclase and the conversion of ATP into cAMP.
Describe the G-alpha t signaling pathway.
Photons induce conformation change in rhodopsin, causing conformational change in G-alpha t (transducin). G-alpha t activates cGMP-specific phosphodiesterase, which catalyzes the conversion from cGMP into 5’GMP. Reduced cellular cGMP levels causes cation channels to close, reducing intracellular sodium and calcium levels. Reduced calcium levels activates receptor guanylyl cyclase, which catalyzes the conversion from GTP into cGMP. Increased cGMP levels causes the cation channel to reopen and cell to depolarize.
There are seven members in the receptor guanylyl cyclase family, of which 5 exist in the human genome. GC-A and GC-B are natriuretic peptide receptors. GC-C is found to be important in GI regulation (linaclotide). GC-D and GC-F are not found in the human genome, and GC-E and GC-F are photoreceptors in the retina.
Rhodopsin-transducin signaling is involved in adjustment to light. Patients taking PDE5 inhibitors may experience blue-field and increased susceptibility to blindness as a result of off-target effects to PDE6, the predominant isoform in the retina.
Describe the G-alpha q signaling pathway.
G-alpha q mediates phospholipase C-beta signaling. Agonist binding to the alpha-1 adrenoreceptor activates the G-alpha q subunit, which activates phospholipase C beta, catalyzing the conversion of PIP2 into diacylglycerol and IP3. Increased diacylglycerol levels activate protein kinase C, activating effector proteins and downstream transcription. Increased IP3 levels mediate calcium release from the ER, increasing intracellular calcium levels, activating the Ca-calmodulin pathway.
G-alpha q signaling can also activate GEF (guanine exchange factor), which catalyzes the conversion of GDP-RhoA to GTP-RhoA and activates downstream effectors, causing the stabilization of actin filaments, promoting adhesion and proliferation.
Protein isoform phospholipase C gamma is involved in mediating receptor tyrosine kinase signaling. PLC-gamma is recruited by phosphorylated EGFR and catalyzes the cleavage of PIP2 into DAG and IP3.
Describe the G-alpha 12 signaling pathway.
Agonist binding induces receptor conformational change, leading to the activation of G-alpha 12, which activates GEF (guanine exchange factor). GEF can also be activated by G-alpha q and ROS. GEP converts GDP-RhoA into GTP-RhoA, which is associated with the stabilization of actin filaments, promoting adhesion, actomyosin contractility, and proliferation.
How does receptor regulation occur (especially internalization)?
The hydroxy-rich intracellular C-terminal is phosphorylated by GRK (GPCR kinase). The phosphorylated terminal recruits beta-arrestin, which mediates the internalization of GPCR via clathrin-mediated endocytosis. If the phosphate groups are removed by phosphotases, the GPCR-containing endosomes are trafficked back to the membrane to recycle the receptor. If phosphate groups are not removed, the endosomes are acidified and receptors degraded by the lysosome.
Explain how nuclear receptors work and why it is difficult to design nuclear receptor agonists.
Nuclear receptors are specialized transcription factors. They either exist in the nucleus and cytoplasm at resting. The DNA binding domain is bound by heat shock proteins, which act as molecular chaperones to ensure transactivation does not occur randomly. Once an agonist binds the receptor, it is activated and forms homo/ heterodimers (according to receptor type) and proceeds to transactivate downstream genes. Since gene expression is involved, the onset of action is relatively slow.
Nuclear receptor agonists are difficult to design because of concerns of overactive gene expression and potential carcinogenesis.
What are class I nuclear receptors? What are some drugs that target them?
Class I receptors are also known as steroid receptors. They are targeted by steroids, including glucocorticoids, mineralocorticoids, estrogen, androgen, and progesterone. At resting, they are bound by heat shock proteins and remain in the cytoplasm. Upon steroid binding, heat shock proteins are released, and receptors form homodiners and migrate into the nucleus to initiate transcription.
Drugs that target class I nuclear receptors include Fulvestrant, which is the latest drug treating breast cancer. Fulvestrant is a selective estrogen receptor degrader, that once bound, promotes the degradation of estrogen receptors, silencing downstream effects. A conventional drug treating breast cancer is Tamoxifen. Apalutimide is a selective androgen receptor antagonist used to treat prostate cancer. While nuclear receptor agonists are difficult to design, Raloxifene, an estrogen receptor agonist, has been used to treat osteoporosis in menopausal women.
What are hybrid class nuclear receptors?
Hybrid receptors are steroid/ retinoid receptors. At resting, some are located in the cytosol, while others in the nucleus. All are specific for their ligands. Upon activation, hybrid receptors form heterodimers with RXR (retinoic x receptor) to initiate downstream transactivation.
What are class II (orphan) receptors?
Orphan nuclear receptors are often targeted by lipids and fatty acids. Many bind multiple ligands, all at low affinity. All are located in the nucleus at resting. Apart from RXR, which forms homodimers with itself, all other orphan receptors heterodimerize with RXR.
Drugs targeting orphan receptors include Rosiglitazone, which targets PPAR-gamma and is used to treat Type II diabetes, and Tretinoin, which targets RAR (retinoic acid receptor) and is used to treat acne.
What are the three main classes of transmembrane receptor enzymes? Why aren’t classical cytokine receptors included?
The three main classes of transmembrane receptor enzymes discussed are receptor tyrosine kinase, receptor serine/ threonine kinase, and receptor guanylyl cyclase. While activation of classical cytokine receptors involve phosphorylation of tyrosine residues, phosphorylation is mediated by JAK, which is not part of the receptor. Classical cytokines have no inherent enzyme activity and are not considered transmembrane receptor enzymes.
What is the principle of action for receptor tyrosine kinases, and what are some drugs targeting them?
Ligand-binding induces receptor dimerization and trans-auto-phosphorylation. Phosphorylated tyrosine residues recruit adaptor proteins with SH2 and PTB domains that mediate downstream functions. Examples of receptor tyrosine kinases include epidermal growth factor receptor (EGFR), platelet-derived growth factor receptors (PDGFR), and VEGFR.
Gefitinib inhibits EGFR and is used to treat EGFR-positive, metastatic lung cancer. Afatinib is an irreversible EGFR inhibitor that forms disulfide bonds and blocks tumor cell signaling. Imatinib targets PDGF receptors and is used to treat leukemia. Ramibizumab (anti-VEGF-mAb) targets VEGF and prevents its binding with VEGF receptors and reduces abnormal angiogenesis. It is used to treat age-related macular degeneration.
Explain how EGFR functions.
Epidermal growth factor binding induces receptor dimerization and phosphorylation, recruiting adaptors (containing phosphotyrosine-recognizing domains). EGF signaling activates the MAPK pathway, promoting proliferation, phopholipase C-gamma, and activates PIK3, which then activates the Akt pathway, promoting survival.
Describe how TGF-beta receptors function and Smad signaling.
TGF-beta receptors consist of Type I and Type II homodimers. Type II receptors dimers are constitutively phosphorylated (and active). Upon binding with TGF-beta, Type II receptors migrate to Type I receptor dimers, oligomerize, and phosphorylate the serine/ threonine residues on Type I receptors. The phosphorylated serine/ threonine residues recruit R-smads (binding via MH2 domain), which couple with co-Smads and migrate into the nucleus to initiate transcription (DNA-binding via MH1). Smad signaling is negatively regulated by I-smads, which can be recruited by the phosphorylated serines and threonines but lack the MH1 (DNA-binding) domain to transactivate. TGF-beta is involved in many complex, and even contradicting pathways, so it is hard to design targeted therapies. Available drugs include Luspatercept and Sotatercept, which are activin receptor ligand traps.
Explain what Luspatercept and Sotatercept do.
Luspatercept and Sotatercept are activin receptor ligand traps. They are structurally analogous with with activin receptors but cannot confer its biological function. Luspatercept is an activin receptor type IIB fusion protein that is used to treat anemia in beta-thalassemia. Reduced Smad signaling enhances erythroid maturation. Sotatercept is an activin receptor type IIA fusion protein and used to treat pulmonary arterial hypertension. Reduced Smad signaling reduces the proliferation of endothelial cells, reversing the narrowing og blood vessles.
What are the seven members of the GC family and how are they related to natriuretic peptide signaling?
The receptor guanylyl cyclase family includes 7 members, GC-A to GC-G. The most well-studied receptor guanylyl cyclases are natriuretic peptide receptors. Natriuretic peptides are ligands that promote vasorelaxation, sodium secretion, and cell proliferation. Natriuretic peptide receptors include NPR-A (GC-A), NPR-B (GC-B), and NPR-C (clearance receptor). NPR-A is targeted by ANP and BNP, while NPR-B is targeted by CNP. GC-C is important in GI regulation. Natural ligands of GC-C are guanylin and uroguanylin. One drug targeting GC-C is linaclotide. Binding to GC-C activates its guanlylyl cyclase function, increasing cellular cGMP levels, thereby increasing chloride and bicarnbonate release, while reducing sodium uptake, increasing the osmotic pressure in the GI lumen so to increase water content to treat constipation. GC-D and GC-G are not found in the human genome. GC-E and GC-F are related to photoreceptors in the eye.
What is the function of GC-C? What are its natural ligands, and how does Linaclotide function?
GC-C is related to GI regulation.
What are the three classes of cytokine receptors and briefly explain their features.
The three main classes of cytokine receptors are classical cytokine receptors, TNF receptors, and IF-1 receptors. Classical cytokine receptors consist of type I and type II receptors. Type I receptors (including single-chain, gp-130, beta, and gamma) all contain the wsxws motif. All classical receptors follow the JAK-STAT signaling pathway.
Explain the JAK-STAT pathway
Cytokine binding to classical cytokine receptors, causing conformational change and JAK (close proximity) to cross phosphorylate and phosphorylate cytokine receptors. Tyrosine phosphorylation recruits STATS, which are phosphorylated by JAK and dimerize. STAT dimers migrate into the nucleus and mediate transcriptional activity.
STAT homodimers are negatively regulated by PIAS (protein inhibitor of activated stats). SOCS (suppressor of cytokine signaling) are also expressed at the same time and exit the nucleus to inhibit phosphorylation and mediate ubiquitination of phosphorylated JAK.
Explain TNFR and dual signaling
TNFR2 signaling (recruitment of TRAF1 and TRAF2) is anti-apoptosis and pro-inflammation. Fas receptor (FADD and caspase 8 activation) is pro-apoptosis and anti-inflammation. TNFR1 receptor has dual signaling. Due to dual signaling, it is difficult to design targeted drugs for TNFR. Anti-TNF mAb binds excessive TNF.
Explain IL-1 signaling and its receptor inhibition
Ligand binding induces the dimerization between IL-1RI and IL-1RAcP (accessory protein), and the dimerization recruits MyD88, which then activates downstream pathways and mediate transcription of pro-inflammatory genes.
Negative regulation includes IL-1RII (decoy receptor lacking TIR domain), IL-1Ra binding (natural antagonist), and soluble cytokine-binding proteins that bind excessive cytokines in the extracellular space.
