L15 Overview of Cell Communication

一、Principles of cell communication

Ways of cell communication:

Contact mediated

• display molecules on cell surface
• recognized by receptor on another cell

Non-contact mediated

• chemical signal
• nearby or at a distance

Many bacteria, for example, respond to chemical signals that are secreted by their neighbors and accumulate at higher population density. This process, called quorum sensing, allows bacteria to coordinate their behavior, including their motility, antibiotic production, spore formation, and sexual conjugation

Communication between cells in multicellular organisms is mediated mainly by extracellular signal molecules.

Signals and receptors

A signaling cell: signaling molecules

A responding cell: receptors

1. Steps in cell signaling

The functional process of signaling molecule

1. Synthesis
2. Release by signaling cell
3. Transport to target cell
4. Detection by a specific receptor protein
5. Change by receptor-signal complex (trigger)

2. Type of extracellular signals (form or molecules)

One of the key challenges in cell biology is to understand how a cell reacts to and integrates different signals in order to make decisions: to divide, to move, to differentiate, and so on…

Origin:

• External signal
• Internal signal

Physical:

• Light
• mechanical force
• Heat

Chemical:

1. water soluble chemical

2. lipid soluble (diffuses across membrane) chemical

• proteins, peptides, amino acid derivatives
• Nucleotides
• steroids, retinoids, fatty acid derivatives
• gases( NO, CO), etc.

Reception of the signals depends on receptor proteins, usually (but not always) at the cell surface, which bind the signal molecule.

The binding activates the receptor, which in turn activates one or more intracellular signaling pathways or systems. These systems depend on intracellular signaling proteins, which process the signal inside the receiving cell and distribute it to the appropriate intracellular targets.

The targets that lie at the end of signaling pathways are generally called effector proteins, which are altered in some way by the incoming signal and implement the appropriate change in cell behavior.

• Depending on the signal and the type and state of the receiving cell, these effectors can be transcription regulators, ion channels, components of a metabolic pathway, or parts of the cytoskeleton

In most cases, however, signaling cells secrete signal molecules into the extracellular fluid. Often, the secreted molecules are local mediators, which act only on cells in the local environment of the signaling cell. This is called paracrine（旁分泌） signaling (Figure 15–2B). Usually, the signaling and target cells in paracrine signaling are of different cell types, but cells may also produce signals that they themselves respond to: this is referred to as autocrine（自分泌） signaling.

A quite different strategy for signaling over long distances makes use of endocrine cells, which secrete their signal molecules, called hormones, into the bloodstream. The blood carries the molecules far and wide, allowing them to act on target cells that may lie anywhere in the body

3. Diverse receptors

Many signal molecules act at very low concentrations (typically ≤ 10^–8^ M), and their receptors usually bind them with high affinity (dissociation constant Kd ≤ 10^–8^ M; see Figure 3–44).

In most cases, receptors are transmembrane proteins on the target-cell surface

In other cases, the receptor proteins are inside the target cell, and the signal molecule has to enter the cell to bind to them: this requires that the signal molecule be sufficiently small and hydrophobic to diffuse across the target cell’s plasma membrane

Each Cell Is Programmed to Respond to Specific Combinations of Extracellular Signals

• Many cells, for example, require a specific combination of extracellular survival factors to allow the cell to continue living; when deprived of these signals, the cell activates a suicide program and kills itself—usually by apoptosis, a form of programmed cell death
• differentiation into a nondividing state (called terminal differentiation) frequently requires a different combination of survival and differentiation signals that must override any signal to divide.

A signal molecule often has different effects on different types of target cells

• The different effects of acetylcholine (乙酰胆碱) in these cell types result from differences in the intracellular signaling proteins, effector proteins, and genes that are activated. Thus, an extracellular signal itself has little information content; it simply induces the cell to respond according to its predetermined state, which depends on the cell’s developmental history and the specific genes it expresses.

receptor protein, not a signal molecule, provides a response

Most of signals are proteins which are larger or not hydrophobic and cannot go through the membrane.

• They bind to membrane receptors activating intracellular signaling molecules.

Two types of cell signaling receptors, cell-surface receptors and intracellular receptors, which work differently.

Signals impact on effector proteins to alter metabolism, change cell shape or movement or alter gene expression.

The same signals can trigger different effects, depending on the cell type

There Are Three Major Classes of Cell-Surface Receptor Proteins

These cell-surface receptors act as signal transducers by converting an extracellular ligand-binding event into intracellular signals that alter the behavior of the target cell.

1. Ion-channel-coupled receptors, also known as transmitter-gated ion channels or ionotropic receptors, are involved in rapid synaptic signaling between nerve cells and other electrically excitable target cells such as nerve and muscle cells
2. G-protein-coupled receptors act by indirectly regulating the activity of a separate plasma-membrane-bound target protein, which is generally either an enzyme or an ion channel. A trimeric GTP-binding protein (G protein) mediates the interaction between the activated receptor and this target protein
3. Enzyme-coupled receptors either function as enzymes or associate directly with enzymes that they activate (Figure 15–6C). They are usually single-pass transmembrane proteins that have their ligand-binding site outside the cell and their catalytic or enzyme-binding site inside

• There are also some types of cell-surface receptors that do not fit easily into any of these classes but have important functions in controlling the specialization of different cell types during development and in tissue renewal and repair in adults

1. Signal processing can convert a simple signal into a complex response.
   • In many systems, for example, a gradual increase in an extracellular signal is converted into an abrupt, switch-like response.
   • In other cases, a simple input signal is converted into an oscillatory response, produced by a repeating series of transient intracellular signals.
   • Feedback usually lies at the heart of biochemical switches and oscillators, as we describe later.
2. Integration allows a response to be governed by multiple inputs. As discussed earlier, for example, specific combinations of extracellular signals are generally required to stimulate complex cell behaviors such as cell survival and proliferation (see Figure 15–4). The cell therefore has to integrate information coming from multiple signals, which often depends on intracellular coincidence detectors; these proteins are equivalent to AND gates in the microprocessor of a computer, in that they are only activated if they receive multiple converging signals
3. Coordination of multiple responses in one cell can be achieved by a single extracellular signal. Some extracellular signal molecules, for example, stimulate a cell to both grow and divide. This coordination generally depends on mechanisms for distributing a signal to multiple effectors, by creating branches in the signaling pathway.
   • In some cases, the branching of signaling pathways can allow one signal to modulate the strength of a response to other signals

Receptor protein, not a signal molecule, provides a response

Depending on receptor, response may be completely different (e.g., in case of acetylcholine)

• Acetylcholine receptors in heart muscle cells and salivary gland cells are identical.

However, they result in different effect or proteins activation

Signal transduction/relay

Three classes of cell surface receptors

Cell surface receptors rely on intracellular signaling proteins to relay the signal!

Cell-Surface Receptors Relay Signals Via Intracellular Signaling Molecules

Some intracellular signaling molecules are small chemicals, which are often called second messengers (the “first messengers” being the extracellular signals). They are generated in large amounts in response to receptor activation and diffuse away from their source, spreading the signal to other parts of the cell.

Some, such as cyclic AMP and Ca2+, are water-soluble and diffuse in the cytosol, while others, such as diacylglycerol, are lipid-soluble and diffuse in the plane of the plasma membrane. In either case, they pass the signal on by binding to and altering the behavior of selected signaling or effector proteins.

• Many of these proteins behave like molecular switches. When they receive a signal, they switch from an inactive to an active state, until another process switches them off, returning them to their inactive state.

1. second messenger

Signaling pathways often contain two different types of messengers:

cAMP

• The first messenger molecules: extracellular signals (signaling molecule)

• The second messenger molecules:

  • small molecules which are generated in large numbers after the receptor activation.
  • They are either hydrophilic or lipid diffusing:
    • Cyclic nucleotides: cAMP, cGMP
    • Ca2+
    • Diacylglycerol (DAG): modified lipid activates PKC
    • Inositol triphosphate (IP3)
  • Second messenger work on effector proteins and they relay the signals.

cGMP

2. Intracellular signaling pathways

Relay signal forward

• Scaffold, anchor
• Amplify
• Integrate multiple signals
• Distribute signals to more than one effector
• Modulate the activity of signaling proteins

Steps of intracellular signaling pathway:

1. activated by an extracellular signal (binding to a receptor protein)
2. The receptor activates one or more intracellular signaling pathways. (involving a series of signaling proteins)
3. Finally, one or more of the intracellular signaling proteins alter the activity of effector proteins and thereby the behavior of the cell.

Many intracellular signal molecules have active and inactive states

The switch between forms is due to

• Protein phosphorylation (Kinases (serine/threonine or tyrosine) attach phosphate, phosphatases dethatch phosphate)
• GTP-binding
• cAMP or Ca2+ binding
• Ubiquitination, etc.

Intracellular Signals Must Be Specific and Precise in a Noisy Cytoplasm

• The binding of a signaling molecule to the correct target is determined by precise and complex interactions between complementary surfaces on the two molecules
• Another important way that cells avoid responses to unwanted background signals depends on the ability of many downstream target proteins to simply ignore such signals. These proteins respond only when the upstream signal reaches a high concentration or activity level

In these and other ways, intracellular signaling systems filter out noise, generating little or no response to low levels of stimuli

The largest class of molecular switches consists of proteins that are activated or inactivated by phosphorylation (discussed in Chapter 3). For these proteins, the switch is thrown in one direction by a protein kinase, which covalently adds one or more phosphate groups to specific amino acids on the signaling protein, and in the other direction by a protein phosphatase, which removes the phosphate groups

• There are two main types of protein kinase.
  1. The great majority are serine/threonine kinases, which phosphorylate the hydroxyl groups (-OH) of serines and threonines in their targets.
  2. Others are tyrosine kinases, which phosphorylate proteins on tyrosines

Protein phosphorylation by kinases

It is one major way of post-translational modification to regulate protein activity
$>$ 30% of all human genome proteins can be phosphorylated
$>$ 520 human kinases (kinome)
$>$ 150 protein phosphatases

Two categories: Serine/Threonine kinase; Tyrosine kinase

Protein kinases are major therapeutic targets in human diseases:

• e.g. : Acute leukemia:
• Gleevec targets BCR-ABL kinase (Uncontrolled Cell Growth)

GEF and GAP

The other important class of molecular switches consists of GTP-binding proteins (discussed in Chapter 3). These proteins switch between an “on” (actively signaling) state when GTP is bound and an “off” state when GDP is bound.

In the “on” state, they usually have intrinsic GTPase activity and shut themselves off by hydrolyzing their bound GTP to GDP (Figure 15–7B). There are two major types of GTP-binding proteins.

1. Large, trimeric GTP-binding proteins (also called G proteins) help relay signals from G-protein-coupled receptors that activate them (see Figure 15–6B).
2. Small monomeric GTPases (also called monomeric GTP-binding proteins help relay signals from many classes of cell-surface receptors.

Specific regulatory proteins control both types of GTP-binding proteins. GTPase-activating proteins (GAPs) drive the proteins into an “off” state by increasing the rate of hydrolysis of bound GTP. Conversely, guanine nucleotide exchange factors (GEFs) activate GTP-binding proteins by promoting the release of bound GDP, which allows a new GTP to bind.

In the case of trimeric G proteins, the activated receptor serves as the GEF.

Not all molecular switches in signaling systems depend on phosphorylation or GTP binding

• We see later that some signaling proteins are switched on or off by the binding of another signaling protein or a second messenger such as cyclic AMP or Ca2+, or by covalent modifications other than phosphorylation or dephosphorylation, such as ubiquitylation
• Most signaling pathways contain inhibitory steps, and a sequence of two inhibitory steps can have the same effect as one activating step (Figure 15–9). This double-negative activation is very common in signaling systems

3. signaling complexes

One simple and effective strategy for enhancing the specificity of interactions between signaling molecules is to localize them in the same part of the cell or even within large protein complexes, thereby ensuring that they interact only with each other and not with inappropriate partners.

Such mechanisms often involve scaffold proteins, which bring together groups of interacting signaling proteins into signaling complexes, often before a signal has been received

High speed and specificity in signaling is achieved by the formation of “signaling complexes”

Simply bringing intracellular signaling proteins together into close proximity is sometimes sufficient to activate them.

• A signaling complex brings all the different molecules that interact with each other in close proximity. This greatly reduces the time the components would need to “find each other”.
• Induced proximity, where a signal triggers assembly of a signaling complex, is commonly used to relay signals from protein to protein along a signaling pathway.

Signaling complexes can be established in different ways:

• Assembly of the complex via scaffolding proteins on an inactive receptor
  • Can be pre-regulated
  • Specific region
• Assembly of the complex on an activated receptor
• Assembly of the complex at the membrane on phosphoinositide docking sites

Pre-formed “signaling complex”

• Receptor and some intracellular signaling proteins to be activated in sequence are pre-assembled into a signaling complex on the inactive receptor by a scaffold protein.

Assembly of signaling complex on an activated receptor

• A signaling complex assembles transiently on a receptor only after the binding of an extracellular signal molecule has activated the receptor.
• The activated receptor phosphorylates itself at multiple sites, which then act as docking sites for intracellular signaling proteins.

Assembly of signaling complex on phosphoinositide docking sites

• Activation of a receptor leads to the phosphorylation of specific phospholipids (phosphoinositides) in the adjacent plasma membrane.
• These phosphoinositides then serve as docking sites for specific intracellular signaling proteins 1 & 2, which can now interact with each other.

4. Interaction Domains

Induced proximity, where a signal triggers assembly of a signaling complex, is commonly used to relay signals from protein to protein along a signaling pathway. The assembly of such signaling complexes depends on various highly conserved, small interaction domains, which are found in many intracellular signaling proteins.

There are many types of interaction domains in signaling proteins.

• Src homology 2 (SH2) domains and phosphotyrosine-binding (PTB) domains, for example, bind to phosphorylated tyrosines in a particular peptide sequence on activated receptors or intracellular signaling proteins.

• Src homology 3 (SH3) domains bind to short, proline-rich amino acid sequences.

• Some pleckstrin homology (PH) domains bind to the charged head groups of specific phosphoinositides that are produced in the plasma membrane in response to an extracellular signal;

They enable the protein they are part of to dock on the membrane and interact with other similarly recruited signaling proteins

• 它们使它们所属的蛋白能够停靠在膜上并与其他类似募集的信号蛋白相互作用

Some signaling proteins consist solely of two or more interaction domains and function only as adaptors to link two other proteins together in a signaling pathway.

Another way of bringing receptors and intracellular signaling proteins together is to concentrate them in a specific region of the cell

Conserved interaction domains are important for protein binding which modulates intracellular signaling proteins

1. Src homology 2 (SH2) domains and Phosphotyrosine-binding (PTB) domains - Bind phosphotyrosine (p-Y)

2. Src homology 3 (SH3) domains: bind proline rich domains

3. Pleckstrin homology (PH) domains: bind phosphoinositides

5. Signaling Cascade

Many intracellular signaling proteins controlled by phosphorylation are themselves protein kinases, and these are often organized into kinase cascades. In such a cascade, one protein kinase, activated by phosphorylation, phosphorylates the next protein kinase in the sequence, and so on, relaying the signal onward and, in some cases, amplifying it or spreading it to other signaling pathways.

Phosphorylation cascade

Signaling protein itself is a kinase which can phosphorylate and activate downstream effectors

1. The MAP kinase module is activated by Ras.

   • The three-component module begins with the MAP kinase kinase kinase Raf.
   • Ras recruits Raf to the plasma membrane and helps to activate it.

2. Raf activates the MAP kinase kinase MEK, which activates the MAP kinase Erk.

3. Erk in turn phosphorylates a variety of downstream proteins, including other protein kinases, as well as transcription regulators in the nucleus.

The resulting changes in protein activities and gene expression cause complex changes in cell behavior

6. Signal integration

• Coincidence of multiple signals

7. Characteristics of signal transduction

The Relationship Between Signal and Response Varies in Different Signaling Pathways

The function of an intracellular signaling system is to detect and measure a specific stimulus in one location of a cell and then generate an appropriately timed and measured response at another location

1. Response timing varies dramatically in different signaling systems, according to the speed required for the response.

   • In some cases, such as synaptic signaling (see Figure 15–2C), the response can occur within milliseconds.
   • In other cases, as in the control of cell fate by morphogens during development, a full response can require hours or days.

2. Sensitivity to extracellular signals can vary greatly.

   • Hormones tend to act at very low concentrations on their distant target cells, which are therefore highly sensitive to low concentrations of signal.
   • Neurotransmitters, on the other hand, operate at much higher concentrations at a synapse, reducing the need for high sensitivity in postsynaptic receptors.
   • Sensitivity is often controlled by changes in the number or affinity of the receptors on the target cell. A particularly important mechanism for increasing the sensitivity of a signaling system is signal amplification, whereby a small number of activated cell-surface receptors evoke a large intracellular response either by producing large amounts of a second messenger or by activating many copies of a downstream signaling protein.

3. Dynamic range of a signaling system is related to its sensitivity. Some systems, like those involved in simple developmental decisions, are responsive over a narrow range of extracellular signal concentrations. Other systems, like those controlling vision or the metabolic response to some hormones, are highly responsive over a much broader range of signal strengths. We will see that broad dynamic range is often achieved by adaptation mechanisms that adjust the responsiveness of the system according to the prevailing amount of signal.

   ---

4. Persistence of a response can vary greatly. A transient response of less than a second is appropriate in some synaptic responses, for example, while a prolonged or even permanent response is required in cell fate decisions during development.

   Numerous mechanisms, including positive feedback, can be used to alter the duration and reversibility of a response.

5. Signal processing can convert a simple signal into a complex response. In many systems, for example, a gradual increase in an extracellular signal is converted into an abrupt, switch-like response. In other cases, a simple input signal is converted into an oscillatory response, produced by a repeating series of transient intracellular signals. Feedback usually lies at the heart of biochemical switches and oscillators, as we describe later.

6. Integration allows a response to be governed by multiple inputs. As discussed earlier, for example, specific combinations of extracellular signals are generally required to stimulate complex cell behaviors such as cell survival and proliferation (see Figure 15–4). The cell therefore has to integrate information coming from multiple signals, which often depends on intracellular coincidence detectors; these proteins are equivalent to AND gates in the microprocessor of a computer, in that they are only activated if they receive multiple converging signals

7. Coordination of multiple responses in one cell can be achieved by a single extracellular signal. Some extracellular signal molecules, for example, stimulate a cell to both grow and divide. This coordination generally depends on mechanisms for distributing a signal to multiple effectors, by creating branches in the signaling pathway. In some cases, the branching of signaling pathways can allow one signal to modulate the strength of a response to other signals.

Speed of responses: slow or fast response

The Speed of a Response Depends on the Turnover of Signaling Molecules

The speed of any signaling response depends on the nature of the intracellular signaling molecules that carry out the target cell’s response. When the response requires only changes in proteins already present in the cell, it can occur very rapidly

When the response involves changes in gene expression and the synthesis of new proteins, however, it usually requires many minutes or hours, regardless of the mode of signal delivery

Fast response: alter protein function

• Alteration of protein function (i.e. activation/inactivation/modification) is immediate (i.e. seconds or less) upon signal reception, leading to fast responses.

These inactivation processes play a crucial part in determining the magnitude, rapidity, and duration of the response.

Slow response: through transcription/translation

• Changes in gene expression and protein synthesis (i.e. de novo protein) start 1 hour or later after signal reception, resulting in slow responses.

Proteins that have higher turn over rate react to stimuli in a faster manner

• The amount and the activity of signaling molecules are important!
• The importance of fast turnover (synthesis/degradation).

1. high turn over: fast/high synthesis rate and quick degradation
2. low turn over: slow/low synthesis rate and slow degradation

turnover rate can determine the promptness of the response when an extracellular signal arrives.

Many proteins in signaling have short half lives:

• ensures/allows for quicker responses
• allows high frequency of the signals(…similar to the low affinity binding of the receptors)

Many signaling proteins undergo conversion between inactive and active states:

• ensures/allows for quicker responses than de novo protein synthesis

Cells Can Respond Abruptly to a Gradually Increasing Signal

Some signaling systems are capable of generating a smoothly graded response over a wide range of extracellular signal concentrations (Figure 15–15, blue line); such systems are useful, for example, in the fine tuning of metabolic processes by some hormones. Other signaling systems generate significant responses only when the signal concentration rises beyond some threshold value. These abrupt responses are of two types.

1. One is a sigmoidal response, in which low concentrations of stimulus do not have much effect, but then the response rises steeply and continuously at intermediate stimulus levels (Figure 15–15, red line). Such systems provide a filter to reduce inappropriate responses to low-level background signals but respond with high sensitivity when the stimulus falls within a small range of physiological signal concentrations.
2. A second type of abrupt response is the discontinuous or all-or-none response, in which the response switches on completely (and often irreversibly) when the signal reaches some threshold concentration (Figure 15–15, green line). Such responses are particularly useful for controlling the choice between two alternative cell states, and they generally involve positive feedback, as we describe in more detail shortly.

A similar sharpening of response is seen when the activation of an intracellular signaling protein requires phosphorylation at more than one site. Such responses become sharper as the number of required molecules or phosphate groups increases, and if the number is large enough, responses become almost all-or-none

Responses are also sharpened when an intracellular signaling molecule activates one enzyme and also inhibits another enzyme that catalyzes the opposite reaction

Positive and negative feedback

Signaling can cause both, an “all-or-none” response or a “smoothly-graded” response”

“All-or-none” response

Cooperative responses that reach a certain threshold:

• e. g. 4 cAMPs binding to PKA and multiple sites of phosphorylation

Concerted effects that activate one enzyme and simultaneous inhibit another enzyme catalyzing the opposing reaction

Positive feedback can generate an “all-or none” response.

1. Positive feedback gives switch-like response

Positive Feedback Can Generate an All-or-None Response

In positive feedback, the output stimulates its own production; in negative feedback, the output inhibits its own production

Those that regulate cell signaling can either operate exclusively within the target cell or involve the secretion of extracellular signals.

If the positive feedback is of only moderate strength, its effect will be simply to steepen the response to the signal, generating a sigmoidal response like those described earlier; but if the feedback is strong enough, it can produce an all-or-none response

A stimulus activates protein A, which, in turn, activates protein B, while protein B then acts back to increase the activity of A.

Effects of positive feedback

Through positive feedback, a transient extracellular signal can induce long-term changes in cells and their progeny that can persist for the lifetime of the organism.

all cells in a population do not respond identically to the same concentration of extracellular signal, especially at intermediate signal concentrations where the receptor is only partially occupied.

• Positive feedback amplify the signal

Activated E kinase acts back to promote its own phosphorylation and activation!

The signal kinase switches the system from an “off” state to an “on” state. With the positive feedback, the “on” status persists - even after the stimulus has stopped.

Positive feedback converts a short signal in a long-term effects even after the stimulus has been removed (or the signal drops back below its critical value).

• It memorizes the signal!

2. Negative Feedback

Negative Feedback is a Common Motif in Signaling Systems

By contrast with positive feedback, negative feedback counteracts the effect of a stimulus and thereby abbreviates and limits the level of the response, making the system less sensitive to perturbations

+ Such reactions can convert a short-term signal in a long-term answer!

A stimulus activates protein A, which, in turn, activates protein B, while protein B then acts back to increase the activity of A.

• Negative feedback change signal to oscillations

The basal activity of the I phosphatase dephosphorylates (deactivates) the activated E kinase at a steady, low rate.

With a short delay, the system shows a strong, brief response when the signal is abruptly changed, and the feedback then drives the response back down to a lower level.

A delayed negative feedback with a long enough delay can produce responses that oscillate.

With a long delay, the feedback produces sustained oscillations for as long as the stimulus is present, then no further response.

How Negative Feedback

Detects changes of concentration of signals.

There are several ways to achieve these:

Cells Can Adjust Their Sensitivity to a Signal

The target cells accomplish this through a reversible process of adaptation, or desensitization, whereby a prolonged exposure to a stimulus decreases the cells’ response to that level of stimulus. In chemical signaling, adaptation enables cells to respond to changes in the concentration of an extracellular signal molecule (rather than to the absolute concentration of the signal) over a very wide range of signal concentrations.

Receptors can also become inactivated on the cell surface—for example, by becoming phosphorylated—with a short delay following their activation. Adaptation can also occur at sites downstream of the receptors, either by a change in intracellular signaling proteins involved in transducing the extracellular signal or by the production of an inhibitor protein that blocks the signal transduction process.

Some ways in which target cells can become adapted (desensitized) to an extracellular signal molecule

1. Receptor sequestration
2. Receptor down-regulation
3. Receptor inactivation
4. Inactivation of signaling protein
5. Production of inhibitory protein

二、General methods to study cell signaling

co-IP

Applications for co-IPs:

• Protein-protein interactions in general
• To find other protein interaction partners
• To analyze receptor-ligand interaction
• To analyze kinase-substrate interaction

Step:

1. Incubation with an antibody
2. Antibody binds to antigen (target molecules)
3. Protein A-conjugated beads bind to the antibody
4. Sedimentation of beads by centrifugation

Western Blotting

Western blotting (WB) (SDS-PAGE followed by WB and immunodetection)

Western blotting analyzing activation of cell signaling

In vitro kinase assay

Samples:
analysis of the kinase activity of three polo-like kinases (PLKs) using 32P in a substrate (casein) phosphorylation reaction in the absence (-) or presence (+) of the kinase inhibitor BI 2536.

Procedure:

1. Purify protein in vitro (either isolation or production of recombinant proteins)Set up in vitro protein assay with substrates and necessary components such as ATP, etc.Upper panel: 32P-phosphorylated casein
2. Analyze protein activity by comparing signal strength.

shRNA/siRNA-mediated “knock-down” or using inhibitors

sh (short hairpin) RNA /si (small interfering) RNA mediated depletion of specific mRNA via RNA interference (RNAi).

Many enzymes have relatively specific inhibitors.

• Example: cGMP-dependent protein kinase G (PKG-Ia) can phosphorylate VASP and Src upon EGF perception by PKG-I$\alpha$.

Phenotype rescue assay

Application:
To prove that one signaling protein locates upstream or downstream of another.

1. Deletion of A or B leads to a certain defect
2. Expression of activated C can rescue this defect.

Summary

General test for protein-protein interaction

• Protein co-immunoprecipitation (co-IP)
• “pull-down” assays
• SDS-PAGE (Polyacrylamide gel electrophoresis) followed by Western blotting (WB) and immunodetection using specific antibodies
• In vitro kinase assay

RNA interference (RNAi)

• short hairpin (shRNA)/short interfering (siRNA) induced “knock-down”
• Functional analysis using chemical inhibitors
• Rescue analysis of defects
• Life cell imaging analysis and spectro-microscopical analysis

三、Classification of receptors

1. G-protein coupled receptor
   1. via cAMP-PKA
   2. via phospholipids: IP3 and DAG, Ca2+
   3. Via NO
2. Enzyme-coupled receptor
   1. Receptor tyrosine kinase (RTK)
   2. Ras signaling
   3. Rho family GTPases
   4. PI3K/Akt signaling
   5. TGF-$\beta$ superfamily
   6. Cytosolic tyrosine kinase
   7. Cytokine receptor/JAK-STAT signaling
3. Other signaling
   1. Notch signaling
   2. Wnt signaling
   3. Hedgehog signaling
   4. NF$\kappa$B
   5. Circadian clocks
4. Nuclear receptors

GPCRs

G (GTP-binding)-protein-coupled receptors (GPCRs)

The activation of a G-protein-coupled receptor triggers indirectly the activation of another membrane protein (via a G-protein).

• The G-protein “mediates” between the activated receptor and the other membrane protein.
• That’s why this receptor type it is called “G-protein-coupled” receptor.

Enzyme-coupled receptors

These receptors either are enzymes themselves and are activated by the signaling molecule or they associate with enzymes, which they activate.

Nuclear receptors (intracellular receptors)

Nuclear receptors are Ligand-modulated gene regulatory proteins

Ligands of nuclear receptors:

Steroid hormones (made from cholesterol) :

• Cortisol ( secreted from cortex to adrenal gland)
• Sex hormones (estradiol, testosterone, progesterone
• Vitamin D (synthesized in the skin under sunlight)
• molting hormone ecdysone (insects)

Thyroid hormone: (made from tyrosine)

Retinoids ( made from vitamin A)

1. how nuclear receptors work

2. Common features of nuclear receptors

Work either as homodimer of heterodimer

Serve both as ligand receptor and as gene transcription factor

All of these receptors possess 3 functional domains:

• DNA binding domain
• Gene transactivation domain
• Ligand binding domain

Signaling molecules that can cross the membrane

Signaling molecules: small hydrophobic molecules that can cross the plasma membrane.

• Examples:
  • Nitric oxide (NO) gas, carbon monoxide (CO) gas
  • Steroid hormones, thyroid hormones, retinoids, vitamin D, etc.
  • Steroid hormones are small enough (+ hydrophobic) to go through the membrane
  • They bind to receptor proteins which are transcription regulators and may be located in nucleus or in cytoplasm

These signaling molecules bind to an intracellular receptor.

1. Steroid Responses

Cytosol location

• receptor bound to inhibitor
• ligand binding activates receptor
• translocates to nucleus on ligand binding

Nuclear location

• binds ligand and DNA
• becomes transcription factor

Some nongaseous signal molecules that bind to intracellular receptors

• All of them are small and hydrophobic!