UNIT 15 – Cell Surface Receptors (Q&A) | MZO-001 MSCZOO | IGNOU
SAQ 1
a) What would happen if there were substances that could bind to Ga subunits just like GTP does, but could not be hydrolysed by the intrinsic GTPase?
If substances were present that could bind to the Gα subunits just like GTP does, but could not be hydrolyzed by the intrinsic GTPase activity, it would lead to a persistent activation of the G-protein signaling pathway. This is because the normal process of signal termination in G-protein signaling involves GTP hydrolysis by the intrinsic GTPase activity of the Gα subunit. When GTP is hydrolyzed to GDP, the Gα subunit becomes inactive, effectively terminating the signaling cascade.
However, if a substance mimics GTP binding but does not undergo hydrolysis, the Gα subunit would remain in its active GTP-bound form indefinitely. This leads to prolonged activation of downstream signaling pathways, such as the activation of second messengers like cAMP, DAG and IP3, and the continuous activation of effector proteins. Such persistent signaling can result in abnormal cellular responses, as the cell remains in a state of continuous "activation" without returning to a resting state.
This scenario could have multiple consequences:
1. Uncontrolled Cellular Proliferation:
Persistent activation of signaling pathways can lead to uncontrolled cell growth and division, which is a characteristic feature of cancer. For example, in the case of mutations in the G-protein pathway (like the K-ras mutation), cells are continuously activated, leading to the overproduction of signaling molecules that drive abnormal cell proliferation.
2. Altered Cellular Functions:
The prolonged activation of signaling pathways could disrupt normal cellular functions. For example, the activation of the MAPK pathway, which is involved in cell growth, differentiation and survival, might cause an imbalance, leading to pathological conditions.
3. Overstimulation of Effector Proteins:
The unregulated signaling could overstimulate effector proteins like adenylyl cyclase, phospholipase C, or ion channels, which could cause imbalances in cellular activities such as ion flux, enzyme activities and changes in gene expression.
4. Metabolic Imbalance:
In some cases, persistent signaling could lead to metabolic disruptions. For example, the continuous activation of the Gαs protein could cause sustained high levels of cAMP, affecting processes like glycogen breakdown or lipid metabolism.
b) What is the role of RGS proteins?
Regulators of G-protein signaling (RGS) proteins are crucial components in the regulation of G-protein-coupled receptor (GPCR) signaling. GPCRs are involved in the transmission of extracellular signals into cells and are essential for controlling a variety of biological processes, including growth, metabolism, immune responses and neurotransmission. When a ligand binds to a GPCR, it activates the G-protein by promoting the exchange of GDP for GTP on the Gα subunit, causing downstream signaling. However, the cell must be able to terminate this signal appropriately to maintain homeostasis and avoid excessive or prolonged signaling, which can lead to disease states like cancer, cardiac arrhythmias, or neurological disorders. This is where RGS proteins come into play. RGS proteins act as critical regulators that accelerate the deactivation of the G-protein, ensuring that the signal is turned off at the right time. Their main function is to enhance the GTPase activity of the Gα subunit, enabling it to hydrolyze GTP to GDP more quickly, thus shutting off signaling and resetting the system for future signals.
Role of RGS Proteins
1. GTPase-Activating Protein (GAP) Activity:
The primary function of RGS proteins is to act as GTPase-activating proteins (GAPs). They speed up the intrinsic GTPase activity of the Gα subunit, promoting the hydrolysis of GTP to GDP. This accelerates the inactivation of the Gα subunit, ensuring that signaling is quickly terminated. This action is vital for maintaining brief signaling events and avoiding persistent activation of signaling pathways.
2. Signal Termination and Regulation:
RGS proteins are essential for terminating signaling once the signal has been transmitted. By promoting faster GTP hydrolysis, they prevent prolonged activation of downstream pathways. This regulation is crucial for preventing overactivation of signaling in pathways responsible for heart rate, neurotransmitter release and immune responses. It ensures that cells can respond to new signals in a timely manner.
3. Controlling Signal Duration and Intensity:
RGS proteins control the duration and strength of signaling. By turning off signals promptly, they prevent excessive activation of pathways that could lead to overstimulation. This regulation ensures that physiological processes, such as cell movement or muscle contraction, occur only when necessary, thereby maintaining proper cellular function.
4. Preventing Abnormal Activation:
Unregulated G-protein signaling can lead to diseases like inflammation and cardiovascular disorders. By promoting timely deactivation of G-proteins, RGS proteins prevent the prolonged activation of pathways that could result in disease conditions. This ensures that the body’s responses are controlled and appropriate.
5. Specificity in Pathway Regulation:
RGS proteins exhibit specificity toward different Gα subunits, such as Gαi, Gαq, or Gαs, and regulate distinct signaling pathways. This allows RGS proteins to fine-tune responses in different signaling contexts, such as regulating calcium signaling or inhibitory pathways.
SAQ 2
Fill in the blanks:
a) Inhibitory Gi, has the same .................. and ................... subunits as stimulatory Gs, but a distinct .................. subunit
Answer: 𝛃, γ, ɑ (Giɑ)
b) Giɑ•GTP complex on dissociation from G𝛃γ ...................... adenylyl cyclase.
Answer: inhibits
c) Almost all of the various effects of cAMP are mediated by ..................... .
Answer: protein kinase A
d) Binding of cAMP by R subunit occurs in a .................... fashion; as a result, even minor changes in cytosolic cAMP have a sizable impact on kinase activity.
Answer: co-operative
e) A common characteristic of numerous signalling pathways is the rapid activation of an enzyme by the dissociation of ..................... .
Answer: inhibitor
f) PKA operates by phosphorylating a serine or threonine residue that is found within the same sequence motif that is .................... .
Answer: X-Arg-(Arg/Lys)-X-(Se/Thr)-Φ, where X stands for any amino acid and Φ for an amino acid that is hydrophobic.
SAQ 3
What are the different ways in which GPCR regulates cellular functions upon hormone binding?
G-protein-coupled receptors (GPCRs) are a large family of membrane receptors that sense extracellular signals such as hormones, neurotransmitters and sensory stimuli. Upon ligand (hormone) binding, GPCR undergoes a conformational change and activates heterotrimeric G-proteins by promoting the exchange of GDP for GTP on the Gα subunit. The GTP-bound Gα subunit and Gβγ dimer then dissociate and modulate various downstream effectors inside the cell.
GPCRs regulate cellular functions through the following four main mechanisms:
i) By Activating Protein Kinase A (PKA)
Many hormones like epinephrine act via Gs-protein-coupled receptors. The activated Gαs subunit stimulates the enzyme adenylyl cyclase, which converts ATP to cyclic AMP (cAMP). The increased levels of cAMP then activate protein kinase A (PKA). Activated PKA phosphorylates various target proteins, enzymes and transcription factors, leading to diverse cellular responses like increased heart rate, glycogen breakdown and lipolysis.
ii) By Modulating Ion Channels
GPCRs also regulate ion channel activity, especially through the Gβγ subunits. For example, muscarinic acetylcholine receptors in cardiac muscle activate G-proteins that directly open K⁺ channels, slowing down the heart rate. In some neurons, GPCRs regulate Ca²⁺ channels, thereby controlling neurotransmitter release. These rapid effects are important in neuronal signaling and muscle function.
iii) By Activating Phospholipase C (PLC)
Some GPCRs couple with Gq proteins, which activate the enzyme phospholipase C-β (PLC-β). PLC hydrolyzes the membrane phospholipid PIP₂ into two second messengers: inositol trisphosphate (IP₃) and diacylglycerol (DAG). IP₃ increases intracellular Ca²⁺ by releasing it from the endoplasmic reticulum, while DAG activates protein kinase C (PKC). Together, these signals modulate functions such as secretion, smooth muscle contraction and gene expression.
iv) By Regulating Gene Transcription
GPCR signaling pathways can ultimately lead to changes in gene expression. For example, cAMP can activate PKA, which then translocates into the nucleus and phosphorylates the transcription factor CREB (cAMP response element-binding protein). CREB binds to DNA and regulates transcription of target genes. Similarly, signals from DAG and Ca²⁺ via PKC and other kinases can also affect transcription. This regulation is crucial for long-term changes like cell growth, differentiation and memory formation.
SAQ 4
a) Define Adaptor proteins
Adaptor proteins are non-enzymatic intracellular proteins that play a critical role in cellular signaling. They act as linkers, bridges, or scaffolds by connecting cell surface receptors to downstream signaling molecules, facilitating the formation of signaling complexes. Although adaptor proteins do not possess enzymatic activity, they are essential in transmitting signals by binding to specific sites on other proteins. This binding ensures the accurate transmission of signals, which is crucial for regulating various cellular functions, such as growth, differentiation, immune responses and apoptosis. In this way, adaptor proteins play a fundamental role in maintaining cellular communication and ensuring signal pathway specificity.
Most adaptor proteins contain specialized structural domains, including SH2 (Src Homology 2), SH3 (Src Homology 3), PTB (Phosphotyrosine Binding) and PDZ domains, which enable them to interact with specific motifs on activated receptors or other signaling proteins. Their ability to recognize phosphorylated tyrosine or serine/threonine residues makes them indispensable in signaling pathways triggered by receptor tyrosine kinases (RTKs), G-protein coupled receptors (GPCRs) and other membrane receptors. By providing structural organization, adaptor proteins ensure that cellular signals are directed accurately and contribute to the precise control of various cellular processes.
Types of Adaptor Proteins
Adaptor proteins are key regulators in intracellular signaling. They do not have intrinsic enzymatic activity, but they mediate protein-protein interactions to assemble signaling complexes. Although adaptor proteins are not usually classified by rigid types like enzymes, based on their functional roles and domain structures, they can be broadly grouped into the following three categories:
1. Modular Domain-Containing Adaptor Proteins
These adaptors contain specific domains that allow them to interact with other signaling molecules. Common domains include:
- SH2 (Src Homology 2): Binds to phosphotyrosine residues
- SH3 (Src Homology 3): Binds to proline-rich regions
- PTB (Phosphotyrosine-binding domain)
- PDZ domains
Examples:
- Grb2: Has SH2 and SH3 domains, links receptor tyrosine kinases (RTKs) to the Ras-MAPK pathway.
- Crk: Connects tyrosine-phosphorylated proteins with effector proteins.
- Nck: Bridges signaling proteins in actin cytoskeleton remodeling.
2. Scaffold Proteins (Specialized Adaptor Proteins)
These adaptor proteins serve as platforms that bring together multiple components of a signaling pathway. They enhance specificity and efficiency by assembling proteins in close proximity. They usually contain multiple protein-binding domains.
Examples:
- KSR (Kinase Suppressor of Ras): Organizes Raf, MEK and ERK in the MAPK pathway.
- JIP (JNK-interacting protein): Scaffold for the JNK MAP kinase pathway.
- AKAPs (A-kinase anchoring proteins): Anchor Protein Kinase A (PKA) near its substrates.
3. Immune Signaling Adaptors
These adaptors play crucial roles in immune receptor signaling. They mediate downstream responses from receptors such as Toll-like receptors (TLRs) or T cell receptors (TCRs).
Examples:
- MyD88: Central adaptor for TLR and IL-1 receptor signaling.
- TRIF: Involved in TLR3 and TLR4 signaling pathways.
- LAT (Linker for Activation of T cells): Essential in TCR signaling cascade.
Importance of Adaptor Proteins:
Adaptor proteins play a fundamental role in regulating and coordinating cellular signaling pathways. These proteins are crucial for maintaining cellular communication by linking cell surface receptors with intracellular signaling molecules, which in turn regulates a variety of essential cellular functions. Below are some key points regarding their importance:
1. Signal Integration and Specificity:
Adaptor proteins ensure the accurate transmission of signals between cell surface receptors and downstream signaling molecules. By acting as linkers or scaffolds, they facilitate the formation of signaling complexes that promote pathway specificity. This is critical in preventing crosstalk between different signaling pathways, ensuring precise cellular responses.
2. Regulation of Cellular Processes:
Adaptor proteins regulate a wide range of cellular processes, including growth, differentiation, apoptosis and immune responses. Their ability to integrate signals from various receptors (e.g., GPCRs and RTKs) allows them to coordinate essential cellular activities, making them key regulators of normal cell function.
3. Role in Development and Differentiation:
During development and tissue differentiation, adaptor proteins are essential in directing the proper cellular responses to extracellular signals. This helps to ensure proper tissue formation and organogenesis, as well as the maintenance of homeostasis within tissues.
4. Involvement in Disease Mechanisms:
Dysfunction in adaptor proteins is associated with a variety of diseases, including cancer, neurodegenerative disorders and immune system abnormalities. Their involvement in key signaling pathways means that any mutations or disruptions in adaptor protein function can lead to uncontrolled cell growth, impaired immune responses, or misregulated cellular differentiation.
5. Facilitation of Complex Signaling Networks:
Adaptor proteins are integral in organizing and facilitating complex signaling networks that integrate inputs from multiple receptors. They help link different signaling molecules, allowing cells to respond appropriately to a wide variety of extracellular cues, such as hormones, growth factors, and environmental signals.
b) Define GEFs
GEFs means Guanine nucleotide Exchange Factors. These are special types of regulatory proteins that are responsible for the activation of small GTP-binding proteins (also called as GTPases). These small GTPases, like Ras, Rho, Rab, Ran and Arf are molecular switches which are very important in many cellular processes like signal transduction, vesicle trafficking, cytoskeleton regulation and cell cycle control.
GTP-binding proteins always exist in two forms. When they are bound to GDP, they are inactive. When they are bound to GTP, they are active. But GDP does not leave the inactive G-protein by itself because it binds tightly. Here, GEFs play an important role. GEFs help the GTPase protein to release GDP and allow a new GTP to bind. This switching from GDP to GTP turns on the GTPase.
For example, Ras protein is a small GTPase involved in growth factor signaling. When a growth factor binds to its receptor on the membrane, it activates a GEF called SOS (Son of Sevenless), which then activates Ras by exchanging GDP for GTP. Active Ras then starts a signaling cascade that leads to cell division. So, without GEFs, Ras cannot become active.
GEFs are also very specific in their function. Some GTPases have their own set of GEFs, while others depend on specific GEFs for their activation and proper function. For example,
- Ras GTPases are activated by GEFs like SOS (Son of Sevenless), which are involved in growth factor signaling and help regulate cell proliferation and differentiation.
- Rho GTPases use specific GEFs like LARG, which are involved in cytoskeleton remodeling, especially during cell movement and shape changes.
- Rab GTPases have their own GEFs involved in vesicle trafficking, where they help in docking and fusion of transport vesicles with target membranes.
- Ran GTPases have specific GEFs like RCC1 (Regulator of Chromosome Condensation 1), which are mainly involved in nucleocytoplasmic transport and mitotic spindle formation during cell division.
- Arf GTPases have their own GEFs which function in vesicle formation, especially in Golgi and endosomal transport pathways by helping in recruitment of coat proteins.
GEFs do not add GTP directly. Instead, they help the GTPase to release GDP. Once GDP is released, the high level of GTP in the cytoplasm automatically binds. This is a natural exchange process, but GEFs increase the rate of this exchange.
GEFs are very important in regulating signal strength, timing and duration. Any mutation or problem in GEFs can cause diseases, especially cancer, because GTPases like Ras may stay active for too long and cause uncontrolled cell growth.
SAQ 5
How does MAPK get activated?
Mitogen-activated protein kinases (MAPKs) are essential enzymes that play a crucial role in regulating a wide range of cellular activities, such as cell growth, differentiation, apoptosis and responses to stress signals. The activation of MAPK occurs through a complex signaling cascade that involves multiple stages and various proteins working in a precise sequence. There are three main types of MAPK pathways: the ERK (extracellular signal-regulated kinase) pathway, the JNK (c-Jun N-terminal kinase) pathway and the p38 MAPK pathway. Despite differences, these pathways follow a common set of activation steps.
The activation of MAPK involves a sequence of events, starting from the binding of an external signal to a cell surface receptor and ending with the regulation of gene expression in the nucleus. The process can be divided into several stages, as follows:
1. Receptor Activation: The First Step in the MAPK Cascade
MAPK signaling begins when an extracellular signal, such as a growth factor, cytokine, or hormone, binds to a receptor on the cell surface. These receptors are often receptor tyrosine kinases (RTKs), although other types of receptors, such as G-protein-coupled receptors (GPCRs), can also initiate the pathway. Once the ligand binds to the receptor, it undergoes a conformational change, which activates the receptor's intracellular domain. This triggers the activation of downstream signaling molecules, setting the stage for the next steps in the MAPK cascade.
2. Activation of Ras: A Key Molecular Switch
Following receptor activation, the next step is the activation of a small GTPase protein called Ras. Ras is activated when the receptor's intracellular domain activates a guanine nucleotide exchange factor (GEF), which causes Ras to exchange GDP for GTP. This makes Ras active. Ras then acts as a molecular switch, linking receptor activation to the downstream components of the MAPK pathway. The activation of Ras is a crucial step that sets the stage for further signaling.
3. Ras Activates Raf: The First Kinase in the Cascade
Once Ras is activated, it binds to and activates Raf, which is a MAPK kinase kinase (MAPKKK). Raf's role is to phosphorylate and activate the next kinase in the sequence, MEK (MAPK/ERK kinase), which is a MAPKK (MAPK kinase). The activation of Raf is an essential step in the cascade and it helps move the signal further down the pathway. Now, the pathway is ready for the next step: the activation of MEK.
4. Activation of MEK: Preparing for MAPK Activation
With Raf activated, it now phosphorylates and activates MEK. MEK is the MAPKK that is responsible for directly activating MAPK. This activation is achieved when MEK phosphorylates MAPK on two specific residues, which fully activates MAPK. The activation of MEK is a critical point in the cascade, as it sets the stage for the final step: the activation of the MAPK itself.
5. Phosphorylation of MAPK: The Final Activation
Once MEK is activated, it then phosphorylates the MAPK, such as ERK, JNK, or p38, depending on the type of signaling pathway. This phosphorylation activates MAPK and enables it to undergo conformational changes. These changes allow MAPK to translocate from the cytoplasm to the nucleus, where it can exert its effects. This is the final step in the activation process.
TERMINAL QUESTIONS
1. Describe the downstream signalling of GPCRs.
G-protein-coupled receptors (GPCRs) are transmembrane receptors that help the cell to receive signals from the external environment and pass them inside the cell. These signals can be in the form of hormones, neurotransmitters and sensory stimuli like smell or light. When a signal binds to the receptor, it activates the intracellular machinery to start a process known as downstream signalling. This signalling starts after the receptor is activated and ends when the final cellular response begins.
There are five main steps in the downstream signalling of GPCRs, starting from ligand binding and ending with kinase activation.
Step 1: Ligand Binding and GPCR Activation
The first step begins when an external ligand like epinephrine or serotonin binds to the extracellular part of the GPCR. This binding causes a conformational change in the structure of the receptor. Because of this shape change, the cytoplasmic portion of the GPCR becomes able to interact with the nearby heterotrimeric G-protein present inside the cell membrane.
This structural change in GPCR prepares it to activate the G-protein, which is the next step.
Step 2: G-protein Activation
The heterotrimeric G-protein is made of three subunits: α (alpha), β (beta) and γ (gamma). In its inactive state, GDP is bound to the α-subunit. When the activated GPCR comes in contact with the G-protein, it causes the GDP to be exchanged for GTP. This process activates the G-protein. As a result, the α-subunit separates from the βγ dimer. Now both these parts become active and start moving inside the membrane.
In the next step, these activated G-protein subunits will pass the signal forward to target enzymes.
Step 3: Activation of Effector Enzymes
Once the G-protein is activated, the α-subunit or the βγ dimer binds to effector proteins such as:
- Adenylyl cyclase (activated by Gsα and inhibited by Giα), which converts ATP to cyclic AMP (cAMP)
- Phospholipase C (PLC) (activated by Gqα), which cleaves PIP₂ into IP₃ and DAG
- Ion channels, such as K⁺ or Ca²⁺ channels, which are regulated by βγ dimers
These enzymes do not directly cause the final response but produce small signalling molecules called second messengers, which spread the signal inside the cytoplasm.
The next step will explain how these second messengers are generated.
Step 4: Second Messenger Production
The effector enzymes now convert existing molecules into second messengers:
- Adenylyl cyclase converts ATP into cAMP
- Phospholipase C breaks PIP₂ into IP₃ and DAG
- IP₃ causes the release of Ca²⁺ ions from the endoplasmic reticulum
These small second messengers quickly move inside the cytoplasm and amplify the signal by spreading it to different regions of the cell.
These messengers now activate special kinases, which is the final step.
Step 5: Kinase Activation and Signal Amplification
The second messengers activate different protein kinases such as:
- Protein kinase A (PKA), activated by cAMP
- Protein kinase C (PKC), activated by DAG and Ca²⁺
- Ca²⁺/Calmodulin-dependent kinase (CaMK), activated by Ca²⁺
These kinases phosphorylate specific target proteins inside the cell. This causes changes in gene expression, metabolic activity, ion channel opening and other functional responses.
This marks the end of downstream signalling, as the signal is now converted into an actual cellular response.
2. Explain the downstream signalling of RTKs.
Receptor Tyrosine Kinases (RTKs) are special types of transmembrane receptors that help the cell to receive and process external signals like growth factors, insulin and other peptide hormones. These signals control very important cellular processes like cell division, cell growth, metabolism, and survival. When a signal molecule binds to the RTK, it activates a cascade of events inside the cell. This entire process is known as downstream signalling of RTKs.
There are five main steps in the downstream signalling of RTKs. The signal starts from ligand binding and proceeds until intracellular kinases activate target proteins.
Step 1: Ligand Binding and Receptor Dimerisation
The process begins when an extracellular signalling molecule such as epidermal growth factor (EGF) or insulin binds to the extracellular domain of RTK. Once the ligand binds, it causes dimerisation of the two RTK monomers. This means two receptor molecules come together and form a dimer.
This dimerisation brings the intracellular tyrosine kinase domains closer, which is important for the next step.
Step 2: Autophosphorylation of Tyrosine Residues
After dimerisation, the kinase domain of one RTK phosphorylates the tyrosine residues present on the other RTK, and vice versa. This process is called autophosphorylation. These phosphate groups attach to the tyrosine residues in the cytoplasmic region.
These phosphorylated tyrosines now serve as docking sites for specific intracellular signalling proteins that contain special domains like SH2 (Src homology 2) or PTB (phosphotyrosine-binding) domains.
Step 3: Recruitment of Adaptor Proteins
Now, specific adaptor proteins are recruited to the phosphorylated RTK. One important adaptor is Grb2 (Growth factor receptor-bound protein 2). Grb2 binds to the phosphotyrosine using its SH2 domain and also binds to SOS (Son of Sevenless) through its SH3 domains.
Grb2 does not work as an enzyme but it acts like a bridge to pass the signal to the next protein, which is Ras.
Step 4: Activation of Ras Protein
Now SOS, which is a guanine nucleotide exchange factor (GEF), activates Ras, a small GTPase present on the inner side of the plasma membrane. It does this by helping Ras to release GDP and bind GTP. This change activates the Ras protein.
Activated Ras is now ready to pass the signal to the MAPK cascade, which is the next major step in the pathway.
Step 5: Activation of MAPK Cascade
Activated Ras first activates Raf (a MAPKKK), which then activates MEK (a MAPKK), and finally ERK (a MAPK). This is called the MAP kinase cascade. Each step involves phosphorylation and amplification of the signal.
ERK (extracellular signal-regulated kinase), once activated, enters the nucleus and activates specific transcription factors, which finally causes changes in gene expression, cell division, survival and differentiation depending on the original signal.
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