AFM Probing Opioid

Signalosome in Neuroblastoma
Outline
I. Introduction to the G-Protein Coupled Receptors, Mu Opiod
Signalosome Complex and the Filamin A
II. Brief Summary of Literature on the Interaction of Filamin A and the
Opioid Receptor
III. The Motivation of the Study
IV. AFM probing the Opioid Signalosome
The GPCR (G-Protein Coupled Receptor)
 The Mu Opiod Signalosome Complex
- the platform for Opioid ligands exerting their pain-relieving actions by
binding to Mu Opioid receptors (MOPR)
- However, the use of opioid is limited since continuous MOPR
activation can develop tolerance.
 
What is Filamin A (FLNA)?
 What is the function of filamin A in actin cytoskeleton
actin/filamin A network has roles in the mechanical response of cells, changing cell shape, and cell locomotion:

1. Filamin A-deficient human melanoma cells (M2 cells) have impaired

cell locomotion and exhibit circumferential blebbing of plasma
membrane which results from the unstable cortex (Cunningham,
1995). On the other hand, A7 cells, showed normal locomotion and
reduced membrane blebbing.
2. Both the cells (M2 and A7) contain similar amounts of Arp2/3, but M2
cells do not have 3-D network of actin filaments while A7 cells do,
which indicates that Arp2/3 complex alone cannot form 3-D network of
actin filaments (Flanagan, 2001)
3. In human periventricular heterotopica (PH), mutations in filamin A
prevent long-range, directed migration of cerebral cortical neurons to
the cortex (Foxet, 1998).
4. Filamin A and actin filaments accumulate at membrane cortices that are under high membrane
tension where they increase the strength of the cortex, which reduces the Ca+2-influx through
mechanosensitive ion channel proteins (Glogaueret, 1998).

5. When mechanical force was applied on human fibroblasts cells using the fibronectin- or collagen-
coated beads through the b1-integrin, the production of filamin A increased (D’Addario, 2001).

6. In the focal adhesion Filamin A connects integrins with the actin cytoskeleton. (Weis, 2002).
FAP52, a focal adhesion-associated phosphoprotein in FAP52, binds the C-terminal region of filamin
A, and these proteins are colocalized at the stress fiber-focal adhesion junction (Nikki, 2002).

These results clearly indicate that filamin A increase the mechanical stability of plasma membrane
and cortex.

Filamin A can respond to signalling molecules and induce a change in actin assembly.
The binding of these signalling molecules with filamin A is an important step in signal
transduction. These results show that filamin A has multiple functions by interacting with many
kinds of cellular proteins such as membrane proteins and various signalling molecules.
Goal: Understand the function and regulation (signal transduction and trafficking of opioid receptors)

Fact: Receptors require interaction with many other proteins.

(G proteins, protein kinases, arrestins and dynamin)

Problem: What proteins are required for the function and

regulation of the mu opioid receptor (MOPr)?
Solution: investigate the involvement of unknown proteins with
MOPR by yeast two-hybrid screens, using the carboxy tail of the
human MOPr as bait and a human brain library
Result: yielded a number of proteins that seemed to bind to the
MOPr C-tail, specifically the Filamin A.
 
WHY FILAMIN A?

Evidence was obtained that there was indeed protein–protein binding between the C-tail of MOPr and FLNA

1. FLNA was known to be a member of the family of actin-cytoskeleton proteins and evidence that the MOPr
interacts with this system has been lacking, though such an interaction is suspected.
2. Stossel at Harvard discovered a cell culture of human melanoma cells (M2), which lacks the gene for FLNA

human melanoma cell line (M2) = lacking the gene for FLNA
control cell line (A7) = M2 transfected with the gene for FLNA
(M2) + myc-tagged MOPr gene
(A7) + myc-tagged MOPr gene

• This permitted investigations of the interactions of FLNA with MOPr and its consequences.
 Characterization of mechanical response using
optical trap to detect the driving force
AFM Probing the MOPR for internalization of opioid receptors and evaluate
the influence of FLNA coupling.
Detection of antibody-receptor recognition

T7 T7
SHSY5Y neuroblastoma cell
AFMline
probe modified with T7-antibody
can identify the T7-tagged receptor
stably expressed by SHSY5Y

T7-tagged MOPR labelled with primary antibody-T7 and secondary antibody

FluoProbes488 after cell immobilization
Functionalization of AFM tips
antibody
O N O
OH O N
OH O C O C O C H
O C O N O
OH O N
O C O C O C H

MT(PEG4)
CT(PEG12) CH3 CH3 CH3
CH3
CH3 CH3 CH3
+
S S
S S S S Antibody S
S
S S
H H S S
NHS/EDC S solution S
+
Au Thiols Au Au Au
AFM Force
contact approach
Spectroscopy

retract
Typical force curves demonstrating the interaction events

Blocked with free anti-T7

Control Experiments
1. Positive:
Gold-substrate coated with T7
Gold-cantilever coated with T7-antibody

2. Negative: Either the substrate or the cantilever was blocked

A. Gold-substrate coated with T7 (then blocked with T7-antibody)
B. Gold-cantilever coated with T7-antibody (then blocked with T7)

3. Checking Thiol-Gold interaction: Uncoated Gold cantilever scanned on

A. coated substrate and
B. on cell surface
Gold Substrate-T7 and Tip-AntiT7/blocked wT7
Sp
1. Positive Control
1. Positive Control
2B. Negative Control (blocked tip)
2B. Negative Control (blocked substrate)
On cell surface
Check Gold-Thiol interaction
Fixed and unfixed cell/uncoated tip
Fixed and unfixed cell/ Tip-AntiT7
With and without DAMGO
Estimate of Specific binding force between
T7 and anti-T7 via PS analysis

Fs= 213.8 pN Fn= 63.8 pN

PSM on control experiment

Fs= 47.5 pN Fn= 68.6 pN

Absence of Filamin A reduced:
1. downregulation of MOPr
2. desensitization of adenylyl cyclase inhibition
3. G protein activation

1. FLNA is required for the activation by mu opioid agonists of the MAP kinase p38
2. By gene deletion studies, FLNA is in the 24th repeat, close to its C-terminal.
3. FLNA lacking the N-terminal (actin binding domain) is as capable as full length FLNA to restore cells to
control status, suggesting that actin binding is not required.
4. Upregulation of MOPr by morphine in M2 cells, downregulation in A7 cells.

An example of downregulation is the cellular decrease in the number of receptors to a molecule, which
reduces the cell's sensitivity to the molecule.