Signaling at the Surface Lecture 21 Vesicular Traffic reading: Chapter 14 Signaling at the Surface reading: Chapter 15

ER vesicle budding and fusion to form new cisterna retrograde return of ER-resident proteins cysternal progression and maturation secretion – constitutive, regulated vesicle transport to lysosomes to maintain structure endocytosis – recycling, destruction, viral infection

GFP-tagged temperature-sensitive trafficking mutant protein (G-protein from Vesicular Stomatitis Virus)

Ts mutant of VSV G protein Progression through Golgi is tightly coupled to specific glycosylation Ts mutant of VSV G protein

Isolated vesicles from two different strains provide cell-free assay for components necessary for progression from one Golgi cisterna to another

why is coat needed for budding? Tension is proportional to Pressure Difference X Radius LaPlace’s Law T = p*r/2

GDP to GTP exchange

Sec23/Sec24 Sec13 and Sec31 proteins also participating in the coat formation are not shown The coat does not assemble unless there is a specific cargo inside which is recognized by the signal receptor

What can block un-coating? 1. Mutations in Sar1 of ARF 2. Non-hydrolyzable analog of GTP (GDP-g-S)

The process of fusion is mediated by several GTP- or ATP-binding proteins

Anterograde and retrograde transport are mediated by different coat complexes

Cisternal Maturation

unidentified coat COPI - purple clartin - red AP complex- blue

Signaling at the cell surface (chapters 15, 16) Binding (again…) and transmembrane signaling Who are the ligands and who are the receptors? G-proteins and G-protein coupled receptors Visual cascade and Receptor adaptation

There are many types of receptors that regulate differentiation and development as well as every day metabolic functions. The signal comes from the outside, the processes are triggered inside

Why the Kd for ligand binding does not reflect the concentration at which half-saturation of the physiological response takes place? 1. There are many more receptors on the surface than required for full response. 2. The cascade of intracellular reactions often provides a many-fold amplification.

Amplification cascade following activation of b-adernergic receptor see the example of glycogen synthesis/breakdown mechanisms, figs 15-25

Kd = 1.4·10-8 M Total binding has contributions from specific (high-affinity) and many non-specific (low-affinity) binding sites. When 125I - labeled insulin is bound in the presence of 100-fold excess of unlabeled insulin, only non-specific binding will be detected.

Binding properties are assessed in competition experiments Kd = 5·10-5 M natural agonist Kd ~ 6·10-8 M stronger agonist Kd = 3·10-9 M high-affinity blocker Occupancies by the primary substrate (L1) and by the competitor (L2) K1,2 – binding constants (1/Kd)

Identification and Cloning of receptors can be done through expression of cDNA libraries in ‘clean’ cells and then probing their responses with specific ligands. Recall the steps that are needed to prepare and screen a cDNA library 1. 2. 3. …