Proteins & Signaling

Membranes ~ Part II

Maintaining Homeostasis• Cells must communicate with their external

environment• Monitoring external conditions determines

cellular responses• Example – E. coli:

– If the bacteria detects a high concentration of lactose, it synthesizes proteins to import and metabolize lactose

– If it detects a higher concentration of glucose, it it synthesizes proteins to import and metabolize gluose

• Membrane proteins help gather information about the environment

Cellular Communication• In multi-cellular organisms,

communication is more complex• Each cell communicates with

dozens of other cells• Determines:

– When it should grow– When it should differentiate or die– When it should release protein

products needed by other cells

Mechanisms of Cell Communication

Communication Through Contact

Two Types of Membrane Proteins

• The membrane is a barrier • Prevents interchange of materials

– Special channels are needed to transport some materials into & out of the cell

• It also prevents free exchange of information– Special receptors are needed to gather

information• Therefore the cell membrane has 2 major

types of proteins:– Transporters – Receptors

Intrinsic & Extrinsic Proteins

• Intrinsic membrane proteins– Embedded in the lipid bilayer– Some extend through it– Transmembrane proteins

• Extrinsic membrane proteins– Absorbed to the surface of the lipid

bilayer– Can be separated from the lipid bilayer

without destroying the membrane

Transmembrane Proteins

• Intrinsic proteins that extend from one side of the membrane to the other are transmembrane proteins.

• Cells pump ions in and out through their plasma membranes.

• More than half the energy that we consume is used by cells to drive the protein pumps in the brain that transport ions across plasma membranes of nerve cells.

• How can ions be transported across membranes that are effectively impermeable to them?

Ligand Gated Ion Channel

Domains• Many transmembrane proteins have three

different domains:• A hydrophilic domain at the N-terminus

– consists of hydrophilic amino acids – pokes out in the extracellular medium

• A hydrophobic domain in the middle of the amino acid chain– often only 20-30 amino acids long– threaded through the plasma membrane– made of amino acids having hydrophobic side

chains

• A hydrophilic domain at the C-terminus – protrudes into the cytoplasm.

Glycoproteins• Many transmembrane proteins are glycoproteins • Sugar side chains are covalently attached to the

hydrophilic domains that protrude into the extracellular membrane.

• A typical mammalian cell may have several hundred distinct types of glycoproteins studding its plasma membrane.

• Each glycoprotein has its extracellular domain glycosylated with a complex branching bush of sugar residues covalently linked to the asparagine side chains.– Some glycoproteins may have 2 or 3 asparagine-

linked sugar side chains, others may have dozens.

Multi-membrane Spanning Proteins

• Some transmembrane proteins have multiple transmembrane domains.

• Hydrophilic domains alternate with hydrophobic domains.

• The protein chain weaves back and forth between opposite sides of the plasma membrane.

• Called serpentine membrane proteins b/c they are snake-like– A common structure in many serpentine

transmembrane proteins involves 7 hydrophobic domains inserted into the plasma membrane, separated by hydrophilic regions that are looped out alternatively into either the cytoplasm or the extracellular space = 7 membrane spanning proteins

Receptors• Specialized transmembrane proteins that

acquire information from the extracellular space

• Relay this information into the cell through the plasma membrane

• Cell surface receptors act as the antennae of the cell.

• Mammalian cells have wide variety of transmembrane receptors

• Two important types: – Growth Factor Receptors – G Protein Receptors

Growth Factor Receptors• Help the cell determine whether or not it

should grow by binding growth factors• Growth factors may be present in the

medium around the cell – Sometimes called mitogens because they

induce the cell to grow and pass through mitosis

– They are polypeptides, often 50-100 amino acids long.

• When present in sufficient quantity, a growth factor (GF) will stimulate a cell to enter into a round of growth and division.

Specificity of Binding

• GFs bind to cell surface GF receptors. • Each type of GF binds to the extracellular

domain of its own specific receptor – will not bind to receptors for other growth

factors.

• Each type of receptor binds specifically to its own ligand– accommodates the appropriate growth factor

in a lock-and-key fashion

Variety of Ligand: Receptor Pairs

• Other ligands besides growth factors convey signals from cell to cell through intercellular space.

• There are at least several hundred distinct receptor: ligand pairs in our body

• Each devoted to the binding of a distinct extracellular ligand such as a growth factor to its cognate receptor.

• Each ligand originates elsewhere and is secreted by a cell or cells specialized for its release.

Transmembrane Signal Transduction

• The binding of a ligand to its receptor is the beginning of the signalling process.

• How does the interior of the cell learn that the ligand has bound?

• How is this translated into information the cell can use?

• Transmission of information by a protein is a form of signal transduction.

An Overview of Cell Signaling

Structure GF Receptor Proteins

• Outside the cell, they have a ligand-binding N- terminal ectodomain

• Inside is a single membrane-spanning transmembrane domain.

• At their C-termini in the cytoplasm, they have a specialized enzyme domain– This becomes activated whenever the

extracellular domain of the receptor binds a GF ligand.

– In the case of many GF receptors, the cytoplasmic enzyme domain contains protein kinase activity.

Kinases & Signal Transduction

• Kinases are enzymes that attach phosphate groups to their substrates.

• Protein kinases take the gamma-phosphates from ATP and transfer them to protein substrates, resulting in the phosphorylation of the substrate proteins.

• The phosphate groups are attached to the tyrosine side chains of substrate proteins that communicate with or lie near the cytoplasmic domains of the GF receptors.

• These receptors are considered to have protein tyrosine kinase activity to distinguish them from many other protein kinases that are devoted to other signalling functions.

Sequence of GF Signal Transduction

• The GF ligand binds to the extracellular domain of its receptor.

• This activates the tyrosine kinase domain at the other end of the receptor in the cytoplasm.

• The tyrosine kinase becomes active and phosphorylates a series of cytoplasmic substrate proteins.

• These are activated or altered functionally as a consequence of being phosphorylated.

• They then send signals further into the cell that result in the cell growing and dividing.

An External Event

• GF ligand does not need to enter the cell in order for transmembrane signalling to occur.

• All active transmembrane signal transduction occurs while the ligand is still in the extracellular space.

Mechanism of Kinase Activation

• How does the association of GF ligand outside the cell cause tyrosine kinase activation inside the cell?

• Some considerations:– There are many copies of each type of GF

receptor molecule that are displayed on the surface of a given cell.

– These receptor molecules, while tethered in the plasma membrane via their hydrophobic transmembrane domains, diffuse laterally through the plane of the plasma membrane.

Dimerization

• When a GF ligand binds to a single receptor molecule, it encourages the dimerization of the receptor with another receptor molecule floating in the plasma membrane.

• Often the GF ligand itself has two receptor-binding ends– enables it to serve as a bridge between the

two receptors – attracts two receptors– encourages their dimerization– stabilizes the resulting receptor/ dimer pair.

Passing the Message

• Dimerization pulls the cytoplasmic domains of the two receptor molecules closer.

• The tyrosine kinase (TK) of one receptor molecule then phosphorylates the kinase domain of the second receptor molecule

• This phosphorylation results in a steric shift in the 3-dimensional structure of the phosphorylated kinase domain

• This causes its functional activation.

Tyrosine Kinase Receptor Dimers

The Final Steps

• The two kinase domains phosphorylate and thereby activate each other.

• Once they are activated, they phosphorylate nearby cytoplasmic substrate proteins that then pass signals further into the cell.

Phosphorylation Cascade

7 Membrane-spanning Serpentine Receptors

Varied Functions• Receptors on cells of the tongue convey taste. • Hundreds of receptor types in our nose convey

information about odors.• A carotenoid molecule related to vitamin A binds

rhodopsin in the rods and cones of our eyes. – It picks up photons which alters its conformation, and

causes the receptor to which it is bound to release signals into the rod/cone cytoplasm that result in our perception of light.

• Baker's yeast cells communicate their sexual identity to each other by release of polypeptide mating factors that bind this type of receptor

• Epinephrine controls the “flight or fight response”

Exchange of Yeast Mating Factors

A G-Protein Receptor

The Role of Epinephrine• Also known as adrenaline• Released by the adrenal glands above the

kidneys in response to stressful stimuli. • Epinephrine travels through the blood stream and

binds to specific receptors on cells in various tissues throughout the body.

• This results in the mammalian fight / flight reaction.

• This includes:– increased heart rate, – decreased blood flow to gut– increased blood flow to skeletal muscles– increased blood glucose

Tracing One Action• Epinephrine acts at many sites to

produce a wide array of physiologic changes

• One of these is increased blood glucose

• Epinephrine causes liver and muscle cells to break down glycogen and release the resulting glucose into the circulation

• We will trace this one action of epinephrine

How Epinephrine Acts

• Epinephrine binds to its receptor on the surface of a variety of cell types throughout the body.

• This beta adrenergic receptor is a 7 membrane-spanning, serpentine receptor embedded in the plasma membranes of these cells.

• As is the case with the growth factor receptors, the epinephrine ligand is not internalized into the cell.

• While bound for a short period of time to its receptor, epinephrine causes the latter to release biochemical signals into the cell cytoplasm.

The Epinephrine Receptor

• These receptors do not depend upon receptor dimerization to transduce signals across the plasma membrane.

• Instead, single receptor molecules will change their 3 dimensional steric configuration in response to ligand binding.

• This steric shift affects the configuration of the cytoplasmic domains of the receptor (the loops of receptor protein that protrude into the cytoplasm).

Cytoplasmic Signal Transduction

• The receptor communicates with the cytoplasm by stimulating a second protein

• This is known as a G protein (G = guanine)• The G protein normally lies near the

receptor in an inactive, quiet state. • When the receptor is activated by ligand

binding, it pokes the G protein. • The G protein responds by switching itself

on, into an active state. • Once in the active state, the G protein

sends signals further into the cell.

The G Protein is Binary• The G protein remains in the active

state for only a brief period, after which it shuts itself off.

• The G protein's two states (ON or OFF) are determined by guanine nucleotide which it binds– thus the term G protein

• When it is inactive, it binds GDP• When active, it binds GTP.

GTP Binding Activates the Protein

• The resting, OFF form of the G protein sits around with its bound GDP.

• When a ligand-activated receptor pokes it, the G protein releases its bound GDP

• It then allows a GTP molecule to jump aboard.

• The GTP-bound form of the G protein is the active ON state.

• While in the ON state, it releases downstream signals.

Feedback Regulation• After a short period of time

(seconds or less), the G protein hydrolyzes its own GTP back to GDP . . .

• Thus shutting itself off. • This hydrolysis represents a

negative feedback mechanism • Ensures that the G protein is only in

the active, signal- emitting ON mode for a short period of time.

Structure of the G Protein• Composed of 3 subunits: alpha, beta &

gamma• In its inactive OFF state, the 3 subunits

are bound together• The alpha subunit binds the guanine

nucleotide, in this case GDP. • When the beta adrenergic receptor

activates the G protein, the alpha subunit releases GDP,

• then binds GTP, • and falls away from the beta and

gamma subunits.

The Signaling Cascade

• Once GTP is bound, the GTP-bound alpha subunit also loses affinity for the receptor.

• It dissociates from receptor, • moves over and pokes another nearby

protein• the enzyme adenylate cyclase, • which is activated by being poked, • and cyclizes ATP into 3'5' cyclic AMP.

The Second Messenger

• cAMP is a second messenger• After G protein encounters adenyl cyclase enzyme,

the alpha subunit of the G protein hydrolyzes its bound GTP and releases the adenyl cyclase– Thus, the G protein reverts to an inactive OFF signalling

state.– The alpha subunit rejoins the beta and gamma subunits

• Adenyl cyclase, no longer poked by the activated a subunit of the G protein, shuts down – stops making cAMP from ATP

• The whole cycle results in only a brief signaling pulse– the production of several hundred cAMP molecules

Cyclic AMP

Second Messenger Action

• Once made, cAMP molecules act as intracellular glycogen

• The high cAMP concentrations enable A kinase to• phosphorylate and thereby activate an enzyme, that

– activates glycogen phosphorylase, which in turn – breaks down glycogen into glucose-l-phosphate

molecules; and

• phosphorylate glycogen synthase, which – turns it off, – preventing the reconversion of the released glucose to

glycogen.

cAMP Second Messenger System

Effect of cAMP on Blood Glucose

• These two changes together ensure the mobilization of glucose through the breakdown of glycogen stored in the liver.

• A number of other reactions are triggered as well that together contribute to the fight/flight response.

Signal Amplification• There is enormous signal amplification in this

cascade. • A single epinephrine molecule (present at 1O-

10M) may cause the activation of dozens of alpha subunits of proteins.

• Each of these in turn will activate the synthesis of a single adenylate cyclase, and

• each of these in turn will synthesize hundreds of cAMP molecules.

• Each of these in turn can activate a cAMP-dependent kinase that will

• modify hundreds of target molecules in the cell.