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2 nd^ Lesson:
Signaling at the Cell Surface
Extracellular signaling molecules are synthesized and released by signaling cells and produce a specific response only in target cells that have receptors for the signaling molecules.
Some signaling molecules, especially hydrophobic molecules such as steroids, retinoids, and thyroxine, spontaneously diffuse through the plasma membrane and bind to intracellular receptors.
The signaling molecule acts as a ligand, which binds to a structurally complementary site on the extracellular or membrane-spanning domains of the receptor. Binding of a ligand to its receptor causes a conformational change in the cytosolic domain or domains of the receptor that ultimately induces specific cellular responses. The overall process of converting signals into cellular responses, as well as the individual steps in this process, is termed signal transduction.
The human genome encodes several thousand G protein–coupled receptors. These include receptors in the visual, olfactory (smell), and gustatory (taste) systems, many neurotransmitter receptors, and most of the receptors for hormones that control carbohydrate, amino acid, and fat metabolism.
Communication by extracellular signals usually involves the following steps:
(1) synthesis and (2) release of the signaling molecule by the signaling cell;
(3) transport of the signal to the target cell;
(4) binding of the signal by a specific receptor protein leading to its activation;
(5) initiation of one or more intracellular signal-transduction pathways by the activated receptor;
(6) specific changes in cellular function, metabolism, or development; and
(7) removal of the signal, which often terminates the cellular response.
The vast majority of receptors are activated by binding of secreted or membrane-bound molecules (e.g., hormones, growth factors, neurotransmitters, and pheromones). Some receptors, however, are activated by changes in the concentration of a metabolite (e.g., oxygen or nutrients) or by physical stimuli (e.g., light, touch, heat).
Signaling Molecules in Animals Operate over Various Distances
In endocrine signaling , the signaling molecules, called hormones, act on target cells distant from their site of synthesis by cells of the various endocrine organs.
In paracrine signaling , the signaling molecules released by a cell affect target cells only in close proximity. The conduction by a neurotransmitter of a signal from one nerve cell to another or from a nerve cell to a muscle cell (inducing or inhibiting muscle contraction) occurs via paracrine signaling. Many growth factors regulating development in multicellular organisms also act at short range.
In addition to cell-surface receptors and second messengers, two groups of evolutionary conserved proteins function in signal-transduction pathways stimulated by extracellular signals:
GTPase Switch Proteins : These guanine nucleotide–binding proteins are turned “on” when bound to GTP and turned “off” when bound to GDP. Conversion of the active into the inactive form by hydrolysis of the bound GTP is accelerated by GAPs (GTPase-accelerating proteins) and RGSs (regulators of G-protein-signaling) and inhibited by GDIs (guanine nucleotide dissociation inhibitors). Reactivation is promoted by GEFs (guanine nucleotide– exchange factors).
Protein Kinases and Phosphatases: Activation of all cell-surface receptors leads directly or indirectly to changes in protein phosphorylation through the activation of protein kinases or protein phosphatases. Phosphatases, which remove phosphate groups, can act in concert with kinases to switch the function of various proteins on or off. The catalytic activity of a protein kinase itself commonly is modulated by phosphorylation by other kinases, by direct binding to other proteins, or by changes in the levels of various second messengers.
Activation of a single type of receptor, however, often leads to production of multiple second messengers, which have different effects. Moreover, the same cellular response (e.g., glycogen breakdown) may be induced by activation of multiple signaling pathways. Such interaction of different signaling pathways permits the fine-tuning of cellular activities required to carry out complex developmental and physiological processes.
An important mechanism for assuring appropriate cellular responses is desensitization of receptors at high signal concentrations or after prolonged exposure to a signal. The sensitivity of a cell to a particular signaling molecule can be down-regulated by endocytosis of its receptors, thus decreasing the number on the cell surface, or by modifying their activity so that the receptors either cannot bind ligand or form a receptor-ligand complex that does not induce the normal cellular response. Such modulation of receptor activity often results from phosphorylation of the receptor, binding of other proteins to it, or both.
G Protein–Coupled Receptors that Activate or Inhibit Adenylyl Cyclase
All G protein–coupled receptors (GPCRs) contain seven membrane-spanning regions with their N-terminal segment on the exoplasmic face and their C-terminal segment on the cytosolic face of the plasma membrane. The GPCR family includes receptors for numerous hormones and neurotransmitters, light-activated receptors (rhodopsins) in the eye, and thousands of odorant receptors in the mammalian nose.
All G protein–coupled receptors contain seven transmembrane helices and presumably have a similar three- dimensional structure. Studies with chimeric adrenergic receptors, suggest that the long C3 loop between helices 5 and 6 is important for interactions between a receptor and its coupled G protein. Presumably, ligand binding causes these helices to move relative to each other. As a result, the conformation of the C3 loop connecting these two helices changes in a way that allows the loop to bind and activate the transducing subunit.
The signal-transducing G proteins contain three subunits designated, and. During intracellular signaling the and subunits remain bound together and are usually referred to as the subunit. The subunit is a GTPase switch protein that alternates between an active (on) state with bound GTP and an inactive (off) state with bound GDP.
Stimulation of a coupled receptor causes activation of the G protein, which in turn modulates the activity of an associated effector protein like adenylyl cyclase, phospholipase C, PDE or ion channels that produce/degrade 2nd messengers or alter membrane voltage. Although the effector protein most commonly is activated by , in some cases it is inhibited. Moreover, depending on the cell and ligand, the subunit, rather than , may transduce the signal to the effector protein. In addition, the activity of several different effector proteins is controlled by
Gα
γ G γβ Gα
G α. GTP
G γβ G α. GTP
Positive and negative regulation of adenylyl cyclase activity occurs in some cell types, providing fine-tuned control of the cAMP level. For example, stimulation of adipose cells by epinephrine, glucagon, or ACTH activates adenylyl cyclase, whereas prostaglandin PGE1 or adenosine inhibits the enzyme. The receptors for PGE1 and adenosine interact with inhibitory Gi, which contains the same and subunits as stimulatory Gs but a different subunit (Gi ). In response to binding of an inhibitory ligand to its receptor, the associated Gi protein releases its bound GDP and binds GTP; the active complex then dissociates from and inhibits (rather than stimulates) adenylyl cyclase.
cAMP-Activated Protein Kinase A Mediates Various Responses in Different Cells
In multicellular animals virtually all the diverse effects of cAMP are mediated through protein kinase A (PKA), also called cAMP-dependent protein kinase.
Inactive PKA is a tetramer consisting of two regulatory (R) subunits and two catalytic (C) subunits. Each R subunit has two distinct cAMP-binding sites; binding of cAMP to both sites in an R subunit leads to release of the associated C subunit, unmasking its catalytic site and activating its kinase activity. Binding of cAMP by an R subunit occurs in a cooperative fashion; that is, binding of the first cAMP molecule lowers the Kd for binding of the second. Thus small changes in the level of cytosolic cAMP can cause proportionately large changes in the amount of dissociated C subunits and, hence, in kinase activity.
PKA mediates various responses in different cells - alters the transcription of specific genes or the activity of specific proteins - by phosphorylation of serine and threonine residues that occurs within the same sequence motif: X-Arg-(Arg/Lys)-X (Ser/Thr)- , where X denotes any amino acid and denotes a hydrophobic amino acid.
Gi α. GTP G γβ
The epinephrine-stimulated increase in cAMP and subsequent activation of PKA enhance the conversion of glycogen to glucose 1-phosphate in two ways: by inhibiting glycogen synthesis and by stimulating glycogen degradation.
Binding of a single hormone molecule to one receptor molecule can result in the synthesis of at least several hundred cAMP molecules before the receptor-hormone complex dissociates and activation of adenylyl cyclase ceases.
Protein kinases and phosphatases
Are involved directly or indirectly in the signal transduction from cell surface receptors. They can be regulated by second messengers (e.g. cAMP).
Alteration on the transcription of a specific gene
PKA phosphorylates and thus inactivates glycogen synthase, the enzyme that synthesizes glycogen. PKA promotes glycogen degradation indirectly by phosphorylating and thus activating an intermediate kinase, glycogen phosphorylase kinase (GPK), that in turn phosphorylates and activates glycogen phosphorylase, the enzyme that degrades glycogen.
protein is composed of four identical subunits, each containing an IP3-binding site in the N-terminal cytosolic domain. IP binding induces opening of the channel, allowing Ca ions to exit from the ER into the cytosol.
The IP -mediated rise in the cytosolic Ca level is only transient because Ca ATPases located in the plasma membrane and ER membrane actively pump Ca from the cytosol to the cell exterior and ER lumen, respectively. Without some means for replenishing depleted stores of intracellular Ca , a cell would soon be unable to increase the cytosolic Ca level in response to hormone-induced IP. A plasma membrane Ca channel, called the TRP channel or the store-operated channel, opens in response to depletion of ER Ca stores. Depletion of Ca in the ER lumen leads to a conformational change in the IP -gated Ca channel that allows it to bind to the TRP Ca channel in the plasma membrane, causing the latter to open.
Opening of IP -gated Ca channels is potentiated by cytosolic Ca ions, which increase the affinity of these channel receptors for IP , resulting in greater release of stored Ca. Higher concentrations of cytosolic Ca , however, inhibit IP -induced release of Ca from intracellular stores by decreasing the affinity of the receptor for IP. This complex regulation of IP -gated Ca channels in ER membranes by cytosolic Ca can lead to rapid oscillations in the cytosolic Ca level when the IP3 pathway in cells is stimulated.
Diacylglycerol (DAG) Activates Protein Kinase C, which Regulates Many Other Proteins
After its formation by hydrolysis of PIP or other phosphoinositides, DAG remains associated with the plasma membrane. The principal function of DAG is to activate a family of protein kinases collectively termed protein kinase C (PKC).
In the absence of hormone stimulation, protein kinase C is present as a soluble cytosolic protein that is catalytically inactive. A rise in the cytosolic Ca level causes protein kinase C to bind to the cytosolic leaflet of the plasma membrane, where the membrane-associated DAG can activate it. Thus activation of protein kinase C depends on an increase of both Ca ions and DAG , suggesting an interaction between the two branches of the IP3/DAG pathway. The activation of protein kinase C plays a key role in many aspects of cellular growth and metabolism. It also phosphorylates various transcription factors;
A small cytosolic protein called calmodulin , which is ubiquitous in eukaryotic cells, functions as a multipurpose switch protein that mediates many cellular effects of Ca ions. Binding of Ca to four sites on calmodulin yields a complex that interacts with and modulates the activity of many enzymes and other proteins. Because Ca binds to calmodulin
3 2+ 3 2+^ 2+ 2+ 2+ 2+ 3 2+ 2+ 2+ 3 2+ 2+
3 2+^ 2+ 3 2+^ 2+ 3 2+^3 3 2+^ 2+ 2+
2
2+
2+
2+ 2+ 2+
in a cooperative fashion, a small change in the level of cytosolic Ca leads to a large change in the level of active calmodulin.
G Protein–Coupled Receptors that Regulate Ion Channels
Many neurotransmitter receptors are ligand-gated ion channels. However many are G protein–coupled receptors. The effector protein for some of these is a Na or K channel; neurotransmitter binding to these receptors causes the associated ion channel to open or close, leading to changes in the membrane potential. Other neurotransmitter receptors, as well as odorant receptors in the nose and photoreceptors in the eye, are G protein–coupled receptors that indirectly modulate the activity of ion channels via the action of second messengers.
Intracellular Receptors
Small lipophilic molecules like steroids (cortisol, progesterone, estradiol, testosterone), thyroxine and retinoic acid diffuse across plasma membrane and interact with intracellular receptors altering gene expression at transcription or post-transcription level. Long term stimulus (hours to days).
The complex signal-receptor can interact with RNA instead of DNA, changing translation.
2+
O complexo hormona-recetor vai permitir a transcrição de um gene.
TGF Receptors and the Direct Activation of Smads
transforming growth factor (TGF ) signaling generally inhibits cell proliferation. Loss of various components of the signaling pathway contributes to abnormal cell proliferation and malignancy. TGF proteins also promote expression of cell-adhesion molecules and extracellular-matrix molecules.
TGF signals certain types of cells to synthesize and secrete growth factors that can, on balance, overcome the normal TFG-induced growth inhibition.
The most abundant TGF receptor, RIII, is a cell-surface proteoglycan, also called -glycan, which binds and concentrates TGF near the cell surface. The type I and type II receptors are dimeric transmembrane proteins with serine/threonine kinases as part of their cytosolic domains. Binding of TGF induces the formation of complexes containing two copies each of RI and RII. An RII subunit then phosphorylates serine and threonine residues in a highly conserved sequence of the RI subunit adjacent to the cytosolic face of the plasma membrane, thereby activating the RI kinase activity.
The transcription factors downstream from TGFβ receptors in Drosophila and the related vertebrate proteins are called Smads.
Ligand binding : Signaling begins with the binding of a TGFβ superfamily ligand to a TGFβ type II receptor. The type II receptor is a serine/threonine receptor kinase, which catalyzes the phosphorylation of the Type I receptor. Receptor recruitment and phosphorylation: It forms a hetero-tetrameric complex ( type II receptor dimer, + type I receptor dimer) with the ligand. The Type II receptor phosphorylates serine residues of the Type I receptor, which activates the protein. SMAD phosphorylation: The Type I receptor phosphorylates the serine residue of the R-SMAD. Phosphorylation induces a conformational change of the R-SMAD and its subsequent dissociation from the receptor complex and SARA (a protein/the SMAD anchor for receptor activation) CoSMAD binding : The now phosphorylated RSMAD has high affinity for coSMAD (e.g. SMAD4) and forms a complex. Transcription: The phosphorylated RSMAD/coSMAD complex translocates into the nucleus where it binds transcription promoters/cofactors and causes the transcription of DNA by inducing expression of target genes.
Bone morphogenetic proteins (BMPs) cause the transcription of mRNAs involved in osteogenesis, neurogenesis, and ventral mesoderm specification.
TGFβs cause the transcription of mRNAs involved in apoptosis, extracellular matrix, neogenesis and immunosuppression. They are also involved in G1 arrest in the cell cycle.
Cytokines control many aspects of growth and differentiation of specific types of cells.
Once the JAK kinases become activated, they phosphorylate several tyrosine residues on the cytosolic domain of the receptor. Certain of these phosphotyrosine residues then serve as binding sites for a group of transcription factors collectively termed STATs. All STAT proteins contain an N-terminal SH2 domain that binds to a phosphotyrosine in the receptor’s cytosolic domain, a central DNA-binding domain, and a C-terminal domain with a critical tyrosine residue. Once a STAT is bound to the receptor, the C-terminal tyrosine is phosphorylated by an associated JAK kinase. This arrangement ensures that in a particular cell only those STAT proteins with an SH2 domain that can bind to a particular receptor protein will be activated. A phosphorylated STAT dissociates spontaneously from the receptor, and two phosphorylated STAT proteins form a dimer in which the SH2 domain on each binds to the phosphotyrosin in the other.
Because dimerization exposes the nuclear-localization signal (NLS), STAT dimers move into the nucleus, where they bind to specific enhancer sequences controlling target genes. Different STATs activate different genes in different cells.
Some cytokines, such as interferon , are produced and secreted by many types of cells following virus infection. The secreted interferons act on nearby cells to induce enzymes that render these cells more resistant to virus infection.
Another related cytokine, erythropoietin , triggers production of red blood cells by inducing the proliferation and differentiation of erythroid progenitor cells in the bone marrow.
Receptor Tyrosine Kinases and Activation of Ras
The ligands for RTKs are soluble or membrane-bound peptide or protein hormones including nerve growth factor (NGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), and insulin.
Ligand-induced activation of an RTK stimulates its intrinsic tyrosine kinase activity, which subsequently stimulates the Ras–MAP kinase pathway and several other signal-transduction pathways. RTK signaling pathways have a wide spectrum of functions including regulation of cell proliferation and differentiation , promotion of cell survival , and modulation of cellular metabolism.
Ras protein is an intracellular monomeric GTPase switch protein that functions in transducing signals from many different RTKs.
Most RTKs are monomeric, and ligand binding to the extracellular domain induces formation of receptor dimers.
Pathways That Involve Signal-Induced Protein Cleavage
This are essentially irreversible pathways in which a component is proteolytically cleaved.
Notch/Delta pathway
Both Notch and its ligand Delta are transmembrane proteins that participate in a highly conserved and important type of cell differentiation in both invertebrates and vertebrates, called l ateral inhibition , in which adjacent and developmentally equivalent cells assume completely different fates.
Notch protein is synthesized as a monomeric membrane protein in the endoplasmic reticulum, where it binds presenilin 1 , a multispanning membrane protein; the complex travels first to the Golgi and then on to the plasma membrane. In the Golgi, Notch undergoes a proteolytic cleavage that generates an extracellular subunit and a transmembrane-cytosolic subunit ; the two subunits remain noncovalently associated with each other in the absence of interaction with Delta residing on another cell. Binding of Notch to Delta triggers two proteolytic cleavages in the responding cell. The second cleavage, within the hydrophobic membrane-spanning region of Notch, is catalyzed by presenilin 1 and releases the Notch cytosolic segment, which immediately translocates to the nucleus. Such signal-induced regulated intramembrane proteolysis (RIP) also occurs in the response of cells to high cholesterol and to the presence of unfolded proteins in the endoplasmic reticulum.
Wnt Signals Trigger Disassembly of an Intracellular Complex, Releasing a
Transcription Factor
Wnt signals act through two cell-surface receptor proteins: Frizzled (Fz), which contains seven transmembrane helices and directly binds Wnt; and Lrp, which appears to associate with Frizzled in a Wnt signal–dependent manner, at least in frog embryos. Mutations in the genes encoding Wingless, Frizzled, or Lrp all have similar effects on the development of embryos. Frizzled protein bear some resemblance to the G protein–coupled receptors.
The central player in intracellular Wnt signal transduction is called -catenin in vertebrates.
This protein functions both as a transcriptional activator and as a membrane–cytoskeleton linker protein.
In the absence of a Wnt signal, -catenin is phosphorylated by a complex containing GSK3, a protein kinase; the APC protein, an important human tumor suppressor; and Axin, a scaffolding protein. Phosphorylated -catenin is ubiquitinated and then degraded in proteasomes.
In the presence of Wnt, -catenin is stabilized and translocates to the nucleus. There, it is believed to associate with the TCF transcription factor to activate expression of particular target genes, depending on cell type.
