Lecture-11 Cell signaling GPCR, RTK and TGF signaling

GPCR signaling cascade

GPCR signaling execute through secondary messengers

Calmodulin Kinase – a molecular memory device

Olfactory signaling system

GPCRs can associate with same effector

leading to different results

GPCR (Gi) signaling in photoreceptor system

GPCR (Gi) signaling in photoreceptor system

GPCR (Gi) signaling in photoreceptor system

GPCRs control various aspects of life

GPCRs in disease

Signaling through cell

surface enzyme-coupled receptors

Receptor Tyrosine

Kinases (RTKs)

Activation of RTKs through dimerization

Activation of RTKs through dimerization

Downregulation of RTK signaling

Activation of RTKs through dimerization

Transforming Growth Factor (TGF)-beta

signaling pathway

Transducers in

TGF signaling

TGF activation

Control of TGF signaling

Fibrodysplasia Ossificans Progressiva (FOP)

TGF dominant disease

Which specific protein function is responsible for exchanging GDP for GTP in the $G_{\alpha}$ subunit?

Guanine nucleotide exchange factor (GEF) function.

What structural change occurs in a GPCR immediately after a ligand binds?

It undergoes a conformational change where the helices move to allow G-protein interaction.

In the GPCR cycle, what triggers the $G_{\alpha}$ subunit to dissociate from the $\beta\gamma$ complex?

The exchange of GDP for GTP on the $G_{\alpha}$ subunit.

What event causes the $G_{\alpha}$ subunit to re-associate with the $\beta\gamma$ trimeric complex?

The hydrolysis of GTP back to GDP by the $G_{\alpha}$ subunit.

Which part of the signaling pathway is considered unique to GPCRs compared to other pathways?

The cycle of how the trimeric G-proteins ($\alpha$, $\beta$, $\gamma$) function.

How does the physical structure of Protein Kinase A (PKA) differ from PKC and PKG regarding its units?

PKA exists as physically separate regulatory and catalytic units after post-processing.

What secondary messenger binds to the regulatory unit of PKA to release the catalytic units?

Cyclic AMP ($cAMP$).

In Protein Kinase C (PKC), how are the catalytic and regulatory units connected?

They are connected by a flexible linker.

Which two secondary effectors are required to activate Protein Kinase C (PKC)?

Calcium ($Ca^{2+}$) and diacylglycerol ($DAG$).

What specific molecule binds to Protein Kinase G (PKG) to release its catalytic domain for activity?

Cyclic GMP ($cGMP$).

Why is Protein Kinase C (PKC) typically localized just under the cell membrane?

It is anchored via its phosphatidyl serine domain and interactions with diacylglycerol.

What is the primary difference between the proteins Calmodulin and Calmodulin-mediated kinase ($CaMKII$)?

They are two distinct and different proteins.

At what minimum number of bound calcium ions does Calmodulin become active?

Two calcium ions.

How does binding four calcium ions instead of two affect Calmodulin's activity?

It remains active for a longer period of time.

According to the cooperative binding model, how do calcium ions typically dissociate from Calmodulin?

They dissociate in pairs (two at a time).

What state of Calmodulin (2-bound or 4-bound) is more prevalent during high-frequency calcium pulsing in neurons?

The 4-bound state.

Once Calmodulin-mediated kinase is phosphorylated at its specific residues, what is required to maintain its activity?

Nothing; it remains active by itself until dephosphorylated by phosphatases.

In the olfactory system, what type of receptor recognizes odorant molecules?

G-protein coupled receptors (GPCRs).

Which enzyme is activated by the $G_{\alpha}$ subunit in olfactory signaling to produce $cAMP$?

Adenylate cyclase.

What is the function of the chloride channel in olfactory neurons?

It amplifies the depolarization signal by pushing chloride ions out of the cell.

How does calcium contribute to olfactory adaptation or tolerance?

It activates phosphodiesterase (PDE) to downregulate $cAMP$ levels.

What determines the specific smell identified by the brain despite having only about 1,000 types of receptors?

The combinatorial signaling and arrangement of triggered receptors in millions of neurons.

In the human eye, what is the primary role of rod cells?

Sensing the brightness or amplitude of light (on/off).

What is the conformation of retinal in Rhodopsin when it is in the dark?

The 11-cis form.

What conformational change occurs in Rhodopsin when it is hit by light?

It converts from the cis form to the trans form.

Which specific phosphodiesterase is activated in the photoreceptor signaling pathway?

Phosphodiesterase 6 (PDE6).

What happens to the $cGMP$ concentration in a rod cell when light activates Rhodopsin?

The $cGMP$ concentration decreases as it is hydrolyzed to $GMP$.

How does the rod cell membrane potential change in response to light?

It becomes hyper-polarized as sodium/calcium channels close.

In the dark, what neurotransmitter is constantly released by rod cells?

Glutamate.

Why is the photoreceptor signaling pathway considered an 'inhibitory circuit'?

Light stops the firing of the photoreceptor, which then allows the bipolar cell to signal.

Which disease involves the cholera toxin inhibiting the GTPase activity of a specific $G_{\alpha}$ subunit?

Cholera.

Regarding Receptor Tyrosine Kinases (RTKs), which domain is highly conserved among different types?

The intracellular enzymatic (tyrosine kinase) domain.

What is the first physical step in the activation of Receptor Tyrosine Kinases (RTKs) upon ligand binding?

Dimerization of the receptors.

Define the term 'transphosphorylation' in the context of RTK activation.

Each receptor in a dimer phosphorylates the tyrosine residues of its partner.

What two features do $SH2$ domains recognize to ensure specific binding to an RTK?

A phosphotyrosine residue and a specific adjacent amino acid side chain.

In the RTK pathway, what is the role of the SOS protein?

It acts as a RAS-GEF to activate RAS by exchanging GDP for GTP.

What laboratory technique uses energy transfer between fluorophores to study the proximity or activation of proteins like RAS?

FRET (Forster Resonance Energy Transfer).

List the three kinases in the MAPK cascade in their order of activation.

RAF (MAPKKK), MEK (MAPKK), and ERK (MAPK).

Why is MEK (MAP Kinase Kinase) considered a rare type of phosphorylase?

It is a dual-specificity kinase that phosphorylates both threonine and tyrosine residues.

Which protein is responsible for marking RTKs for degradation in the lysosome via ubiquitination?

CBL (an E3 ubiquitin ligase).

How do Protein Tyrosine Phosphatases (PTPs) regulate RTK signaling?

They dephosphorylate the receptor to prevent transducer recruitment.

What is the specific genetic mutation responsible for 97% of Achondroplasia cases?

The $G380R$ mutation in the $FGFR3$ gene.

What is the physiological effect of constitutive $FGFR3$ signaling in Achondroplasia?

It inhibits the proliferation of chondrocytes in bone growth zones.

What type of enzymatic activity is characteristic of $TGF-\beta$ receptors?

Serine/threonine kinase activity.

The term 'SMAD' is a portmanteau derived from which two biological model organisms?

$Drosophila$ (Mad) and $C.\ elegans$ (Sma).

Which class of SMAD proteins is directly phosphorylated by the $TGF-\beta$ Type I receptor?

Receptor-regulated SMADs (R-SMADs).

What is the primary role of SMAD4 in the $TGF-\beta$ pathway?

It acts as a Co-SMAD, forming a complex with R-SMADs to enter the nucleus.

What structural motif at the C-terminus of R-SMADs is the target of receptor phosphorylation?

The $SSXS$ motif.

How is the Nuclear Localization Signal (NLS) in R-SMADs made accessible for transport?

It is revealed through a conformational change triggered by phosphorylation.

Which SMAD class lacks a DNA-binding domain and functions to downregulate signaling?

Inhibitory SMADs (I-SMADs).

In the $TGF-\beta$ pathway, how do Inhibitory SMADs (e.g., SMAD 7) typically work?

They recruit phosphatases or E3 ubiquitin ligases to the receptor complex.

What is the function of the Ski protein in $TGF-\beta$ regulation?

It binds the transcription complex and recruits Histone Deacetylases (H-DACs) to stop transcription.

What characterizes the pathology of Fibrodysplasia Ossificans Progressiva (FOP)?

Soft tissues like muscle and skin are progressively converted into bone.

In FOP, what is the nature of the $ALK2$ receptor activation?

It is ligand-independent, often triggered by inflammation or injury.

Concept: $G_{\alpha}$ subunit

Definition: The GTPase component of a heterotrimeric G-protein that hydrolyzes GTP to GDP to terminate signaling.

Concept: Phosphodiesterase (PDE)

Definition: An enzyme that breaks the cyclic bond in secondary messengers like $cAMP$ or $cGMP$, converting them to $AMP$ or $GMP$.

Term: Chondrocyte

Definition: Cells found in cartilage that are responsible for proliferation and hypertrophy during long bone growth.

What secondary messenger is used in olfactory signaling to open sodium/calcium channels?

Cyclic AMP ($cAMP$).

In the visual system, $cGMP$ levels are _____ in the dark, keeping ion channels open.

High

The $TGF-\beta$ Type II receptor activates the pathway by _____ the Type I receptor.

Phosphorylating

Which R-SMAD is specifically mentioned as a common target in $TGF-\beta$ signaling?

SMAD3.

What specific signal allows the R-SMAD/Co-SMAD complex to exit the cytoplasm and enter the nucleus?

The revealed Nuclear Localization Signal (NLS).

Which protein kinase has its regulatory and catalytic units linked by a flexible chain that is released by $cGMP$?

Protein Kinase G (PKG).

What is the primary difference in the target of PKA versus an RTK?

PKA targets serine/threonine residues, while RTKs target tyrosine residues.

In olfactory signaling, why is the internal concentration of chloride ions unique?

The neuronal cytoplasm has a higher concentration of chloride than the external environment.

How does the brain 'learn' according to the theory involving $CaMKII$?

By sensing the frequency and duration of calcium spikes to maintain kinase activity.

Which enzyme converts $cGMP$ to $GMP$ in the rod cell disk membrane?

Phosphodiesterase 6 (PDE6).

What is the purpose of the ridges in the olfactory epithelium?

To increase surface area for odorant molecules to interact with receptors.

In GPCR signaling, $G_{\beta}$ and $G_{\gamma}$ subunits are known to have their own _____ functions, though not detailed in this lecture.

Signaling

What determines the specificity of an RTK's extracellular domain?

The specific ligand it is evolved to bind.

Which kinase in the MAPK cascade directly phosphorylates transcription factors or other target proteins?

ERK (also known as MAP Kinase).

How does $TGF-\beta$ signaling typically end for a specific R-SMAD protein?

It is dephosphorylated or degraded via ubiquitination to stop transcription.

The $PPXY$ motif in I-SMADs is used to recruit _____.

E3 ubiquitin ligases

Which SMAD protein possesses both an NLS and an NES (Nuclear Export Signal)?

SMAD4 (Co-SMAD).

In the $RAS/MAPK$ pathway, the 'M' in MAPK stands for _____.

Mitogen

Under what lighting condition is a rod cell considered to be in its 'dark state'?

When $cGMP$ levels are high and ion channels are open.

What is the common result of activating the MAPK pathway regarding cell behavior?

Stimulation of cell proliferation.

A single olfactory neuron expresses exactly _____ set of olfactory receptors.

One

Which GPCR-related disease is characterized by a persistent inhibitory signal that manifests as a cough?

Whooping cough (Pertussis).