Lecture 2, Oct 11 Important points from 10/7: Ligands and Receptors Ligand-receptor binding shows great sensitivity. Endocrine, paracrine, autocrine, membrane-bound Hydrophilic ligands bind to cell surface receptors Cell surface receptors: G protein coupled; ion-channel linked; receptor tyrosine kinase linked; receptors with intrinsic enzymatic activity Second messengers: inside the cell—effector molecules of cell signaling Signaling: protein kinases; GTP-binding proteins with GTPase activity can function as molecular switches; integration of multiple signals; internalization; protein-protein interactions using adapter proteins.

Signal Transduction: focus on G-proteins and the PKA pathway Many cell-surface receptors are coupled to trimeric signal-transducing G proteins. Trimeric: composed of three different subunits G proteins: bind either GTP or GDP Ligand binding to a G protein coupled receptor activates the associated G protein which in turn activates an effector enzyme to generate an intracellular second message.

All G-protein coupled receptors have 7 membrane spanning regions with their amino termini on the extracellular face and their carboxy termini on the cytoplasmic face of the plasma membrane.

Structure of an inactive G protein—alpha and gamma covalently attached lipid molecules; alpha is GDP bound. Structure of transducin, the G protein in visual transduction

Taste receptors Mu opioid receptor Ion channel on pain fibers

Illustration: binding of epinephrine/norepinephrine to the b adrenergic receptor Mediates the body’s response to stress (e.g. going to the dentist) --release of glucose and fatty acids from liver and fat cells --increased contraction of cardiac muscle Binding of epinephrine to b adrenergic receptor increases the intracellular concentration of cAMP. How does this happen?

cAMP is synthesized within cells from ATP by the enzyme adenylate cyclase. cAMP is degraded by the enzyme cAMP phosphodiesterase

--The b-adrenergic receptor mediates the induction of epinephrine-initiated cAMP synthesis. --Different receptors utilize a common adenylate cyclase (i.e., each receptor does not have its own intrinsic adenylate cyclase).

Martin Rodbell, Nobel Prize 1994 GTP is required for the ligand-induced stimulation of adenylate cyclase. Overall: need, 1) a receptor, 2) a transducer (G-protein) and 3) an amplifier (adenylate cyclase) that generates large amounts of a second messenger.

--Binding of the ligand to the receptor changes its conformation, causing it to bind to the trimeric Gs protein in such a way that GDP is displaced from Gas and GTP is bound. --The Gas-GTP complex dissociates from the Gbg complex, then binds to and activates adenylate cyclase. --activation is short-lived: GTP bound to Gas hydrolyzes to GDP in second, leading to the association of Gas with Gbg and inactivation of adenylate cyclase.

Disassembly of activated G-protein produces TWO signaling components.

Switching off of the G-protein alpha subunit by hydrolysis of its bound GTP

Cholera toxin: modifies Gas by adding an ADP ribosyl group Cholera toxin: modifies Gas by adding an ADP ribosyl group. This modified Gas can bind GTP but cannot hydrolyze it. As a result, there is an excessive and nonregulated rise in intracellular [cAMP].

Activating mutations in Gas underlie fibrous dysplasia, where see excess fibrous growth, which calcifies over time Note: inactivating mutations in Gas underlie another disease, pseudohypoparathyroidism

To review: G protein activation and complex formation are part of a cycle

The Gas stimulates adenylate cyclase; another Ga subunit, Gai, inhibits adenylate cyclase.

Degradation of cAMP is also regulated through the hydrolysis of cAMP to 5”-AMP by cAMP phosphodiesterase. Many drugs affect cAMP phosphodiesterase—including caffeine.

How does increased cAMP activate the cAMP-dependent protein kinase, PKA?

The catalytic subunit of PKA can phosphorylate substrates on serine or threonine residues. It has substrates in the cytoplasm and the nucleus. In the nucleus, PKA can activate transcription of genes containing cAMP response elements, or CREs in their promoter. A specific transcription factor, the cAMP response element binding protein, CREB, binds to this sequence and activates transcription of downstream genes. When CREB is unphosphorylated, it is inactive; only in its phosphorylated state does CREB activate transcription.

How ligand binding to a cell surface receptor can induce gene expression

How ligand binding to a cell surface receptor can induce gene expression

Animation: see http://www.whfreeman.com/lodish/ Lodish book, 5th edition, chapter 14, animation on extracellular signaling. Or Alberts et al., 4th edition, interactive disk

Important points: G protein coupled receptors: receptors with 7 membrane spanning domains. Ligand binding produces signaling to second messenger by binding to and transducing its signal to a trimeric G protein G protein has 3 subunits: a, b and g. Ligand-bound receptor interacts with G protein, causing conformational change. Ga subunit exchanges GDP for GTP and dissociates from Gbg. Both a and b/g can be active signaling components. GTP-bound Ga subunit now associates with and activates adenylate cyclase, which produces cAMP, a second messenger. Gas activates adenylate cyclase, Gai inhibits adenylate cyclase. Intrinsic GTPase activity of Ga terminates signaling.

cAMP activates PKA by binding to the regulatory subunits of the kinase cAMP activates PKA by binding to the regulatory subunits of the kinase. PKA is a tetrameric kinase: 2 regulatory and 2 catalytic subunits. When cAMP binds the regulatory subunits, the catalytic subunits translocate into the nucleus where they can phosphorylate substrates. These can include transcription factors such as CREB and thereby result in changes in gene expression.