Lecture 8: Cell Signaling Principles, G protein coupled receptors

Cell Signaling Principles

• Cell communication mediated by Extracellular Signal Molecules

• Receptor Molecules: senses/bound by receptor molecules, typically at cell surface

  • binding activates receptor —> activates intracellular signaling: proteins or second messengers carry/convey signals to other molecules

• Effector Proteins: are at the end of the pathway and are activated/deactivated/modified to perform function.

  • ex. metabolic enzyme (alters metabolism), transcription regulatory protein (alters gene expression), cytoskeletal protein (alters cell shape/movement).

4 Ways of Intercellular Signaling

• Contact Dependent: cells have to be in direct membrane-membrane contact

  • intimate convo

• Paracrine: chemical molecules act on neighboring cells, signals locally

  • discussion with friends

• Synaptic: performed by neurons, carries signal along axons and release neurotransmitters at the synapse

  • fast, hardwired transmission

• Endocrine: signal molecules called hormones are released into the bloodstream which is then carried almost everywhere in the body, allowing them to act on target cells.

  • public broadcast

Many of the signaling molecules used are the same, its a matter of speed and selectivity in which signals are delivered to targets.

Each Cell is Programmed to Respond to Specific Combinations of Signals

• cells are exposed to hundreds of various signals, however they respond selectively

• Cells require signals to survive, in absence of signals they go through apoptosis

• Various signals are integrated by cell to decide what to do

• Same signal can cause different responses depending on what cell is programed to do

Types of Signals and Receptors

Signals can be

• Water Soluble/ insoluble

  • proteins/ small peptides: growth factors

  • Small molecules: amino acids, fatty acids, nucleotides, steroids, retinoids

  • Dissolved gases: nitric oxide, carbon monoxide

Receptors

• Receptors bind/ recognize signal with high specificity

• Most receptors are transmembrane proteins that bind to extracellular signals, ligands

• There are also intracellular receptors that bind to small hydrophobic molecules that diffuse through the membrane

3 Major Classes of Cell-Surface Receptors

• Ion Channel Couples Receptors

  • aka ligand gated ion channels or ionotropic receptors

  • small number of neurotransmitters transiently open or close an ion channel

• G Protein coupled receptors

  • signal through a gtp binding protein to activate membrane-bound enzymes or ion channels

  • mediates interaction between receptor and target protein

• Enzyme-coupled receptors:

  • are either enzymes themselves or associated with enzymes that become activated upon ligand binding; most are protein kinases - phosphorylate proteins

Intracellular Signaling Molecules Relay the Signals

• Secoond Messengers: small chemicals (cyclic AMP, Ca2+) are generated at/near the receptor during activation, diffusing and binding to downstream proteins, spreading signal

  • 1st signal = extracellular signal

Scaffolding Proteins

• Scaffolding can increase signaling specificity/speed

• Interactions between intracellular signaling molecules need to be very specific and robust in a sea of other molecules/interactions

• This process is aided by co-localizing molecules into signaling complexes by scaffold proteins

  • can be pre-assembled or assembled at/near receptor after activation by phosphorylation

  • activation can cause phosphorylation of nearby phosphatidylinositols (PI) to PIP, which then recruit further signaling proteins that can be activated.

Molecular Domains Mediate Interactions between Signaling Proteins

• Interaction Domains: areas on many signaling proteins that specifically recognize structural motifs on other proteins

  • SH2 and PTB domains bind to phosphorylated tyrosines

  • SH3 domains bind to proline-rich sequences

  • PH domains bind to PIPs

Combining Signaling Events into circuits or networks and their resulting properties

• Signaling Cascades: transform the signal in important and necessary ways

  • composed of signal processing, amplification, integration, and corrdination

  • Signals from more than one source can be integrated, requiring both

• Signal sensitivity, response timing, persistence, and dynamic range vary greatly in different signaling systems

  • response over a range of signal concentrations can be gradual or abrupt

Positive Feedback Loop

• Output of a step causes more of its own production

• All or nothing response pattern or sigmoidal

• Response is persistent, “off” to “on”, and “on” state is self-sustaining,

  • Bistable: when system is self sustaining, doesn’t need continuous stimulus

  • Useful for transient (brief) signal to cause stable long-term changes

Negative Feedback

• output inhibits its own production

  • Short delay dampens response

  • Long delay can cause oscillations

• Causes adaptation or desensitization to the signal, reduced response over time

G-Protein-Coupled Receptors

• largest family of cell-surface receptors

• recognize/bind a wide variety of

  • ligand-hormones

  • Neurotransmitters

  • Proteins/small peptides

  • All molecules that we smell and taste

  • Photons of light

• A common 7 transmembrane alpha helical structure

• Activates Heterotrimeric G proteins, GTP binding proteins

Heterotrimeric G proteins relay signals from GPCRs

• 3 protein subunits: alpha, beta, gamma, anchored in the membrane

• alpha subunit is the GTPase, bound to GDP when inactive

• When a ligand binds to the inactivated GPCR it is then activated and then the alpha subunit of the G protein binds to the GPCR.

  • The GDP on the alpha subunit dissociates and is replaced by a GTP, which then results in the alpha subunit to separate from the beta and gamma subunits.

  • The separated subunits can then interact with downstream signaling proteins such as effectors

• Hydrolysis of the GTP in the alpha subunit turns it off

  • This can be stimulated with interactions with the target proteins

G Protein Regulation of the production of Cyclic AMP

• cyclic amp is a second messenger in some pathways

  • produced by adenylyl cylases from ATP

  • destroyed by cAMP phosphodiesterases

• Some GPCRs are coupled to:

  • Stimulatory G protein, Gs, activates an adenylyl cyclase

  • Inhibitory G protein, Gi, inhibits adenylyl cyclases

cAMP activates PKA in most cells

• cAMP activates cyclic-AMP-dependent protein kinase (PKA)

  • PKA phosphorylates a number of target proteins

• some of cAMP/PKA rewsponses are changes in gene expression which turns on genes

Ca2+ is a ubiquitous second messenger, it’s everywhere

• Ca2+ acts as a secondary messenger in many signaling pathways (not just GPCRs):

  • muscle cells, nerve cells, secretory cells

• some signals cause influx of Ca2+ through the plasma or ER membrane

Calmodulin/ CaM kinases mediate Ca2+ responses

• Calmodulin: is an abundant protein hat is allosterically activated by Ca2+

  • Calmodulin binds and activates Ca2+/ calmodulin dependent kinases (CaM kinases), Cam kinase II

  • 12 subunits, stacked ring structure, one of more activated by calmodulin, which auto phosphorylates to stay active

G Proteins Directly or Indirectly Regulate Ion Channels

• heart muscle receptors —> beta gamma subunits open K+ channels —> inhibitory effect

• Olfactory receptors (smell): G protein —> cAMP opens cAMP gated channels —> results in action potential

• Vision: rhodopsin GPCRs are activated by light —> alpha subunit activates cGMP phosphodiesterase which degrades cGMP

  • Without signaling, higher neuron firing rate

  • In light, cGMP degraded, lower firing rate

Nitric Oxide (NO) gas mediates signaling between cells

• dissolved nitric oxide relaxes smooth muscle cells that constrict blood vessels

• Nitric Oxide synthase produce nitric oxide from arginine

• NO can diffuse through membranes to neighboring cells

• NO activates guanylyl cyclase, which produces cGMP

GPCR Pathway amplification/desensitization

• many components/ enzymes. 2nd messengers in the pathway provide opportunities for signal amplification

Desensitization

• GPCR activates leads to activation of GPCR kinases (GRK), which phosphorylate the receptor and cause binding of arrestin, preventing signaling through G Proteins