Download Chem*3560 Lecture 33: Membrane receptors and signalling

Chem*3560
Lecture 33: Membrane receptors and signalling
Complex organisms indicate developmental or metabolic states
by release of signalling molecules or hormones from one
tissue to stimulate a response in another tissue.
Some of these signalling molecules are relatively nonpolar and
can pass through the bilayer. Steroids like estradiol or
testosterone enter cells and bind to soluble receptor proteins
in the cytoplasm. The complex is imported into the nucleus
where the receptor acts to regulate gene expression.
In animals, more polar signalling molecules such as
epinephrine bind to specific receptors on the exterior
of the plasma membrane. This is also the case for larger
signalling molecules such as the peptide hormones
insulin and glucagon. These receptors are
transmembrane proteins, and presence of the ligand
bound to the exterior is sensed by the cytoplasmic domain
to pass the signal on to the cell interior.
Insulin receptors, as well as the receptors for a variety of cell growth factors, are transmembrane
proteins with a ligand binding domain on the extracellular side and a tyrosine kinase domain on the
cytoplasmic side. Glucagon and epinephrine receptors have seven transmembrane helices, and regulate
adenylate cyclase on the cytoplasmic side.
The insulin receptor tyrosine kinase
The insulin receptor tyrosine kinase consists of a
dimeric structure with two α subunits containing the
insulin-binding domains, and two β subunits with
extracellular domain, a single transmembrane helix and
a cytoplasmic tyrosine kinase domain, which terminates
in an activation domain that contains tyrosine. When the
receptor binds insulin, a reorientation of the
cytoplasmic domains occurs and this allows activation
by autophosphorylation of as many as three tyrosines
near the C-terminal end of each β subunit. (More
accurately, this is mutual cross-phosphorylation within
the dimer) (Lehninger p.445).
The unphosphorylated loop containing Tyr occupies the substrate site, but is constrained so that
phosphorylation can't occur in the absence of insulin. Once activated, the catalytic site becomes
available for the tyrosine kinase to phosphorylate other target proteins.
Insulin has growth regulating and metabolic effects on cells
Insulin behaves much like a variety of tissue specific growth factors, for example, fibroblast growth
factor, epidermal growth factor etc. Each has a unique receptor so that a particular cell type only
responds to its own growth factor. In many cases, the receptor is monomeric in the absence of ligand,
and only forms dimer in the presence of ligand. Proximity due to dimer formation then allows the
cross-phosphorylation of each others tyrosines.
Once the receptor activates its intracellular tyrosine kinase, the growth stimulating signalling pathway
inside the cell follows common elements. The insulin receptor phosphorylates a protein called insulin
receptor substrate, IRS-1 at up to seven tyrosine sites. The phosphorylated IRS-1 binds to and
stays closely associated with the insulin receptor phosphotyrosines.
The signal that results from activated tyrosine kinase is often communicated through a series of
protein-protein binding interactions. Animal cells contain a variety of proteins that contain related
domains called SH domains which stands for Src Homology. These share sequences similar to
domains in Src, one of the first tyrosine kinases to be discovered. (The name Src derives from its
discovery in cells infected with the tumour-inducing chicken sarcoma virus.) SH1 is the tyrosine kinase
catalytic domain itself. SH2 domains bind tightly to phosphotyrosine in another protein. SH3
domains bind a specific proline rich sequence found in certain intracellular signalling proteins.
When IRS-1 becomes phosphorylated, a
SH2-domain protein Grb2 binds to IRS-1's
phosphotyrosines. Grb2 is an adapter protein
whose function is to hold other proteins together.
(This a role similar to TRADD, which links up tumor
necrosis factor receptor to procaspase 8 in the
apoptosis pathway). Grb2 also has two SH3 domains,
which exist in a complex with a protein called Sos
(Son of sevenless - don't ask why, it's too long a
story). Because of IRS-1 is also bound to insulin
receptor, these interactions concentrate Sos in
proximity to the inside of the plasma membrane
(Lehninger p.446).
Here Sos can meet a lipid anchored protein called Ras (first found associated with Rat sarcoma virus).
Since the insulin receptor signalling pathway regulates cell growth, it's not surprising that some of the
factors are implicated with tumour induction.
Ras is a GTP-activated signalling molecule that controls cell proliferation
Ras is a member of a group of small regulatory GTPase proteins
(230 amino acids) which are themselves regulated by binding GTP.
Members of this family regulate a variety of cellular processes, e.g.
Ran - nuclear transport; Rac - vesicle traffic, Rho - actin cytoskeleton.
Ras binds GTP and hydrolyses it very slowly (turnover number 1.2
hr-1, which is 106 times slower than a typical enzyme). Ras passes on
an "active" signal when it contains GTP, and an "inactive" signal
when it contains GDP. The slow GTP hydrolysis serves as a
timing mechanism, and determines how long Ras stays in its
active state. Ras binds GDP very tightly so is unable to release the
product, so remains in the "inactive" signalling state. Sos plays the role
of a guanine nucleotide exchange factor or GEF. When Sos
binds Ras, it allows Ras to exchange the tightly bound GDP for a new
molecule of GTP, thus switching it from an inactive to active state.
Finally Ras is the activating factor for a
Ser-Thr protein kinase called Raf (Ras
activated factor). Raf initiates a
serine-threonine kinase cascade (MAP
Kinase, mitogen associated protein kinase)
which passes on into the nucleus and
activates transcription factors, in particular
for genes associeted with cell
proliferation.
In addition, Raf serves as a GTPase
accelerating protein or GAP, and
speeds up the GTPase rate of Ras to a
respectable 20 s-1 (105 fold speed up).
Before Raf binds, slow GTP hydrolysis
keeps Ras active, giving it a good chance
to meet up with Raf. Fast GAP-induced
hydrolysis of GTP rapidly turns off the Ras "active" signal as soon as Ras has done its job.
This combined effect of a specific GEF (guanine exchange factor) to turn on the signal and a GAP
(GTPase accelerating protein) to turn it off again is a mechanism shared by many of the other small
regulatory GTPases.
Metabolic regulation by insulin receptor
IRS-1 also stimulates another enzyme calle PI3K, phosphotidyl inositol-3 kinase, which adds another
phosphate to the membrane phospholipid phosphatidyl inositol- 4,5-bisphosphate making it into
phosphatidyl inositol-3,4,5-trisphosphate (PIP3 ). This serves as an activator of PIP3 -dependent
protein kinase (PDK), which initiates protein kinase cascades that activate glycogen synthase and
glucose transport (Lehninger p.447).