Cell Signaling Proteins

How do cells transmit extracellular signals to generate intracellular responses?

• Signal transduction refers to the biochemical mechanism responsible for transmitting extracellular signals across the plasma membrane and throughout the cell.

• Receptor protein activation by ligand binding results in one or more of three biochemical responses: (1) covalent modification of target proteins, (2) protein conformational changes, and (3) altered rates of protein expression.

• A signaling pathway is a linked set of biochemical reactions that consists of upstream events occurring at or near the plasma membrane and downstream events that alter the activity or level of target proteins.

• The number of signaling genes in the genome of an organism reflects the need for complex networks of communication within and between individuals.

• Hormones are biologically active compounds that bind to receptor proteins and function as first messengers through endocrine, paracrine, or autocrine mechanisms.

• The functional role of second messengers is to amplify receptor-generated signals that are initiated by a single receptor binding event; many types of second messengers are generated by catalytic enzymes with high turnover rates such as adenylate cyclase, guanylate cyclase, and nitric oxide synthase.

• Receptor activation of the enzyme phospholipase C (PLC) leads to the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP₂), which generates the second messengers diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP₃). In turn, activity of these molecules leads to increased intracellular levels of calcium ions (Ca²⁺), another second messenger.

• Higher eukaryotes contain five abundant classes of receptor proteins: (1) gated ion channels, (2) G protein–coupled receptors, (3) receptor tyrosine kinases, (4) tumor necrosis factor receptors, and (5) nuclear receptors. All but the nuclear receptors are transmembrane proteins that bind extracellular ligands.

• The nicotinic acetylcholine receptor is an Na⁺–K⁺ gated ion channel that transmits physiologic signals across neuromuscular junctions in response to the release of its ligand acetylcholine, a neurotransmitter, from nearby neurons.

• Negatively charged amino acids line the vestibules on the extracellular and intracellular sides of the nicotinic acetylcholine receptor to attract Na⁺ and K⁺. Nonpolar amino acids lining the channel prevent stripping of the ion hydration layers to ensure that the hydrated Na⁺ and K⁺ ions only pass through the open channel in response to acetylcholine binding.

How do G protein–coupled receptors use heterotrimeric G proteins to transmit cell signals initiated by glucagon or epinephrine?

• G protein–coupled receptors (GPCRs) contain seven transmembrane α-helices that are oriented with the N terminus in the extracellular space and the C terminus in the cytoplasm.

• GPCRs transmit extracellular signals to the cytoplasm through direct interaction with a membrane-bound protein complex called a heterotrimeric G protein, which consists of one each of Gα, Gβ, and Gγ subunits (Gαβγ).

• Gα is a member of the G protein family of signaling proteins, which contain an intrinsic GTP hydrolyzing activity (GTPase) that converts the active GTP-bound protein into the inactive GDP-bound protein.

• GPCR-mediated activation of the associated heterotrimeric complex occurs when the GDP bound to the Gα subunit is replaced by GTP, resulting in dissociation of the Gα subunit from the heterotrimeric complex.

• The human genome contains multiple Gα, Gβ, and Gγ genes, encoding proteins that combine to form ~1,100 unique Gαβγ complexes, many of which are involved in sensory perception (sight, smell, taste).

• GPCR activation by ligand binding results in a conformational change in the receptor cytoplasmic domain that facilitates binding of the GDP-bound Gαβγ complex and subsequent stimulation of GDP–GTP exchange in the Gα subunit and dissociation of the Gαβγ complex into Gα–GTP and Gβγ signaling complexes.

• The dissociated membrane-associated Gα–GTP subunit activates several target proteins depending on the specific Gα subtype; the membrane-bound Gβγ complexes also activate target proteins.

• Glucagon and epinephrine signaling in liver cells is mediated by GPCRs, which stimulate downstream signaling through shared and parallel pathways.

• Glucagon binds to glucagon receptors in liver cells to initiate a Gsα-mediated response that stimulates adenylate cyclase, resulting in cAMP activation of protein kinase A (PKA) and net glucose export.

• Epinephrine (adrenaline) binding to β₂-adrenergic receptors activates the same Gsα-mediated pathway as glucagon (shared pathway), whereas epinephrine binding to α₁-adrenergic receptors activates a Gqα-mediated pathway that stimulates the activity of phospholipase C and net glucose export (parallel pathway).

• Guanine nucleotide exchange factors (GEFs) promote GDP–GTP exchange and activation of G protein signaling, whereas GTPase activating proteins (GAPs) stimulate the intrinsic GTPase of G proteins and inhibit G protein signaling.

• The GEF proteins in GPCR-mediated signaling are the ligand-activated receptors themselves, and the GAPs are members of the regulator of G protein signaling (RGS) family of signaling proteins.

• GPCRs are removed from the plasma membrane and recycled to terminate signal transduction. This mechanism involves phosphorylation of the GPCR cytoplasmic tail by G protein–coupled receptor kinases (GRKs) and binding of β-arrestin protein to these phosphorylated residues.

How do receptor tyrosine kinases trigger downstream pathways that control cell growth and metabolism?

• Receptor tyrosine kinases (RTKs) often function as homodimers that bind extracellular ligands. Ligand binding activates a cytoplasmic tyrosine kinase function.

• RTK-mediated autophosphorylation of tyrosine residues in the cytoplasmic tail generates phosphotyrosine binding sites for adaptor proteins, which link downstream signaling pathways to activated receptors.

• Epidermal growth factor receptors (EGFRs) are RTKs that form homodimers upon binding of two epidermal growth factor (EGF) molecules, thereby activating the intrinsic tyrosine kinase activity in each receptor subunit and autophosphorylation of multiple tyrosine residues.

• SH2 domains are adaptor modules in signaling proteins that bind selectively to phosphotyrosine residues in target proteins.

• GRB2 is an SH2-containing adaptor protein that links the Ras signaling pathway to EGFR activation through binding of another adaptor protein, called SOS, to the SH3 domains in the GRB2 protein. SH3 domains bind to proline-rich sequences in target proteins.

• Ras is a G protein that is similar to the Gα subunit of heterotrimeric G proteins and is characterized by three features: (1) it is attached to the cytoplasmic face of the plasma membrane by a lipid anchor; (2) it is activated by GEFs such as SOS; and (3) it is deactivated by stimulation of its intrinsic GTPase activity by GAPs.

• Ras activation initiates a downstream signaling pathway, which consists of a phosphorylation cascade mediated by kinases in the mitogen-activated protein (MAP) family of signaling proteins. This results in gene regulation and increased rates of cell division.

• Oncogenes are cancer-causing, mutated copies of normal genes. Although they were initially discovered as “hitchhiker genes” in animal tumor viruses, they were later shown to be present in a variety of human cancers.

• Many oncogenes encode signaling proteins that contain dominant mutations (gain of function). For example, Ras proteins that are defective in GTPase activation, due to missense mutations in codon 12, stimulate cell division in the absence of growth factor signaling.

• Insulin binds to the insulin receptor, a disulfide-linked RTK. The insulin receptor consists of a transmembrane α₂β₂ complex that is activated by a single insulin molecule binding to one of the α subunits; this mode of ligand binding is an example of negative cooperativity.

• Insulin receptor signaling initiates two downstream signaling pathways: one signals through the Ras G protein to activate the MAP kinase phosphorylation cascade, and the other activates the PI3K pathway, leading to glucose uptake.

• In the PI3K pathway, insulin receptor substrate-1 (IRS-1) protein binds to phosphotyrosine residues on the insulin receptor cytoplasmic tail through a phosphotyrosine binding (PTB) domain, resulting in the tyrosine phosphorylation of IRS by the insulin receptor and subsequent recruitment of PI3K through its SH2 domain.

• In the MAP kinase pathway, Src homology collagen (Shc) protein binds to phosphotyrosine residues on the insulin receptor cytoplasmic tail through a PTB domain, resulting in the tyrosine phosphorylation of Shc by the insulin receptor and subsequent recruitment of the GRB2 adaptor protein through its SH2 domain.

• PI3K phosphorylates PIP₂ to produce PIP₃, which functions as a membrane-docking site for phosphoinositide-dependent kinase-1 (PDK1) through its pleckstrin homology (PH) domains. Phosphatase and tensin homolog (PTEN) converts PIP₃ back into PIP₂ to terminate PI3K signaling through PDK1.

• PDK1 phosphorylates and activates the kinase Akt, which leads to Akt-mediated responses in liver cells that lower blood glucose levels through increased glucose import and liver glycogen synthesis.

How does tumor necrosis factor receptor signaling determine whether a cell survives or initiates programmed cell death?

• Binding of tumor necrosis factor-α (TNF-α) to the trimeric TNF receptor initiates two opposing pathways: one that leads to programmed cell death (apoptosis) and the other that promotes cell survival. The net cellular response to TNF-α signaling is determined by the relative abundance of downstream signaling proteins.

• TNF receptor signaling is mediated by the assembly of an adaptor protein complex, which consists of signaling proteins that share protein binding modules called death domains (DDs) and death effector domains (DEDs).

• TNF receptor–induced cell death involves recruitment of the adaptor proteins TRADD and FADD, leading to the activation of caspase 8 and initiation of a downstream proteolytic cascade that activates caspase 3—the executioner protease.

• The TNF receptor–mediated cell survival pathway involves recruitment of the adaptor kinases TRAF2–NIK and RIP to the receptor complex, which stimulates a phosphorylation cascade leading to increased expression of anti-apoptotic genes.

• Caspase enzymes function as heterotetramers that catalyze proteolytic cleavage of peptide bonds on the carboxyl side of aspartate residues by using a nucleophilic cysteine residue located in the enzyme active site.

How do nuclear receptors function as ligand-activated transcription factors?

• Nuclear receptors are ligand-activated transcription factors that control a wide range of physiologic responses, as governed by (1) ligand bioavailability, (2) cell-specific expression of nuclear receptors and coregulatory proteins, and (3) accessibility of target gene DNA sequences in chromatin to nuclear receptor binding.

• Nuclear receptors bind to specific DNA sequences in target genes by using two zinc finger protein folds. The receptors recruit coregulatory proteins (coactivators or corepressors) that modulate transcriptional initiation rates by modifying chromatin-associated proteins.

• There are two major types of nuclear receptor proteins, which are categorized on the basis of their mode of DNA binding: (1) steroid receptors bind as head-to-head homodimers to inverted repeat DNA sequences, and (2) metabolite receptors bind as head-to-tail heterodimers to direct repeat DNA sequences.

• Peroxisome proliferator–activated receptors are metabolite receptors that selectively bind dietary unsaturated fatty acids and form heterodimers with retinoid X receptors, which are metabolite receptors that bind 9-cis-retinoic acid.

• Glucocorticoids are steroid hormones that are synthesized in the adrenal glands. They bind to the glucocorticoid receptor and regulate a variety of cellular responses, including inflammation, lung cell development, and carbohydrate metabolism.

• The pharmaceutical drugs prednisone, triamcinolone, and dexamethasone are glucocorticoid agonists that function as anti-inflammatory agents by activating glucocorticoid receptor signaling in target cells, leading to increased expression of the annexin I gene and decreased expression of the cyclooxygenase-2 gene.