Download Protein-blot analysis of receptor-ligand interactions

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BIOCHEMICAL SOCIETY TRANSACTIONS
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Received 10 October I987
Protein-blot analysis of receptor-ligand interactions
JONATHAN M. GERSHONI
Department of Biophysics, The Weizmann Institute of Science,
Rehovot 76100, Rehovot, Israel.
Protein blotting was originally introduced as a solid-phase
immunoassay (Gershoni & Palade, 1983; Towbin & Gordon,
1984). However, this technique can also be employed for the
analysis of ligand-receptor interactions. In principle, a
protein sample containing a receptor is resolved by gel
electrophoresis and then blotted to an immobilizing matrix,
such as a nitrocellulose membrane filter, as would be performed for immunoblotting. The blot is subsequently reacted
with the corresponding ligand rather than a solution containing a particular immunoglobulin. The ligand-receptor complex is then detected, usually via autoradiography when the
probe can be radioactively labelled.
Whereas this corollary to blotting appears to be straightforward, there are a number of basic points that can jeopardize the chances of obtaining good results. Ligand overlay
requires that binding capacity be maintained in the blotted
counterpart. In immunodetection, the probe is a large, intact,
native protein complex designed to recognize a distinct and
small immobilized determinant. In the case of receptor
detection, the probe might be extremely small and affinity
and selectivity are expected of the protein which has undergone gel electrophoresis and blotting. These procedures are
usually denaturative and so the dissociation and unfolding of
the receptor components might readily destroy any quaternary and tertiary configurations required for the integrity of
the ligand binding site.
Another issue which should be considered is the detectability of the probe. Where iodinated ligands can be
obtained, demonstration of a complex on the blot is easy.
Many ligands, however, which are quite adequate for binding
assays, are
or lJC-labelled and are thus less convenient
for our purpose.
Nonetheless, in spite of all the above apprehensions, there
are quite a few systems for which protein blotting and ligand
overlay have been very useful (for recent reviews see
Gershoni, 19876, 1988). A case in point is the nicotinic
acetylcholine receptor ( AChR).
"
H
-
The nicotinic acetylcholine receptor
This receptor is a multimeric glycoprotein complex
( a , P y S ) situated on the post-synaptic side of the neuro-
muscular junction. The receptor functions as a ligandregulated ion-channel and by binding the agonist, acetylcholine, it elicits membrane depolarization which leads to
muscle contraction. The antagonist, a-bungarotoxin (BTX),
Abbreviations used: AChR. acetylcholine receptor; BTX.
a-bungarotoxin.
competes for the acetylcholine-binding site with an affinity
of K,= l o - " M. a-Bungarotoxin is a short peptide (74
amino acid residues), derived from snake venom and is easily
iodinated (Popot & Changeux, 1984).
Protein blots have been prepared from crude membrane
preparations containing the AChR. These were then probed
with '251-labelled-BTX and autoradiographed. Only the asubunit of the AChR was found to bind BTX although the
affinity of the complex formed (Kd= lo-' M ) was markedly
reduced as compared with that of intact receptor. Systematic
analysis of the BTX binding to blotted a-subunits has
allowed the precise identification of the toxin-binding site.
Protein blots of proteolysed a-subunits were probed with a
variety of different reagents. The toxin was used to reveal the
fragment containing the ligand-binding site. Concanavalin A
was employed to detect the presence of residue Asn-14 1, the
only asparagine which can be N-glycosylated. Sequencespecific antibodies prepared against synthetic peptides were
used for the final identification of the various proteolytic
fragments generated (Gershoni et al., 1983; Wilson et ul.,
1984; Neumann etal., 1985, 1986).
In addition, protein blotting has been used to determine
the disulphide arrangement of the a-subunit. The AChR was
alkylated with a biotinylated maleimide (Bayer et al., 1985)
and the SH-containing fragments derived from such receptors were identified using I -51-labelled-avidin overlay of blots
(Mosckovitz & Gershoni, 1988).
Protein blotting has been extremely important in the study
of the structure and function of the native AChR. Currently
this approach is being used for the study of bacterially
expressed BTX-binding sites as well (Gershoni et al., 1987a).
Once the binding site within the a-subunit had been identified, subclones of the complementary DNA of the a-subunit
were prepared using the bacterial expression vector pATH2.
Escherichia coli transformants produced a fusion protein
which binds BTX. Subjecting these clones to recombinant
DNA manipulation has allowed the binding-site to be
characterized and the specific contribution of individual residues is being ascertained.
Most exciting is the fact that the bacterially expressed
binding sites have proven to have possible clinical value.
These 'mimic binding sites' act as molecular decoys ('decoyants') and can intercept the toxin in the blood stream. Mice
have been injected with the decoyant or a placebo and then
challenged with neurotoxins. Those which received the
decoyant have a 3-4-fold better chance of survival as
compared with the controls.
Thus by systematic blot analysis of the AChR, not only
has the binding site been mapped, this study has led to a
novel concept for possible drug design, namely: decoyance.
The success of this research has largely been due to the
amenability of the BTX/AChR system to blot procedures.
Some practical considerations of ligand overlay are discussed with the hope that this may be helpful.
1988
624th MEETING, DUBLIN
134,
I’ructicul cotisiderutiori.~
In testing whether o r not a particular system can be subjected to blot analysis a numbcr of preliminary experiments
and parameters should be taken into account.
(i) Dot blot. Before attempting a gel-blot, dot-blotting
should be performed. In doing so, the conditions are determined for an optimal signal-to-noise ratio. In essence, the
protein applied as a dot should contain an intact receptor.
Probing such dots allows the verification of the suitability of
the probe and detection system. Other factors such as the
quench solution and washing conditions can be established.
(ii) Sunnple prepurutiori. In the dot-blot experiment the
receptor is left unperturbed. Most electrophoresis systems
require SDS denaturation of the sample. However. a good
amount of ‘over-kill‘ is often exerted. The sample should not
be boiled, if possible detergent concentration should be kept
to a minimum (0.1%) and the effect o f reducing agents
should be cvaluated.
(iii) Gel electrophoresis. A point which may have an effect
is the time course of running the gel. We often want to obtain
results as fast as possible and thus run gels at high currents
causing unnecessary joule-heating. Running the gel at lower
currents is preferable. In addition, usc o f lithium dodecyl
sulphate and running the gel at 4°C may be helpful.
(iv) Blottirg. Thc buffer composition in blotting may have
effects on the conformation o f the protein being blotted.
Methanol should be avoided as this tends to ‘fix’ the protein.
Whereas electroblotting is used routinely in our studies,
blotting by diffusion might provide time for the sample to
renature if such renaturation is possible. As to renaturation,
pretreatment of the gel before blotting and post-treatment of
the blot prior to probing can be beneficial.
(v) Processing. The affinity of a ligand for a blotted receptor constituent may be much less than that observed for the
native system. Therefore, washing in ice-cold buffer and
reducing processing times might be necessary. Once the
ligand is bound and excess probe is removed some sort of
fixation or cross-linking can be attempted.
J.G. thanks Rachel Samuel for preparing this manuscript. This
research has been supported by the Yeda Foundation. the Israel
Society for Psychobiology, the Minerva Foundation and the Muscular Dystrophy Foundation.
Bayer, E. A,, Zalis, M. G. & Wilchek, M. (1985, Ancrl. Hioc.hetn.
149,520-536
Gershoni, J. M. & Palade. G. E. ( 1983) Arid. Hioc.hrm. 131. I - I5
Gershoni, J. M., Hawrot, E. & Lentz, T. L. ( 19x3) /’roc. Nut/. Acud.
Sci. U.S.A.80,4973-4977
Gershoni, J. M. ( 1 9 8 7 ~ )/’roc. Nut/. Accrd. .Sci. (1.S.A. 84.
43 18-432 1
Gershoni. J . M. ( 19X7h I in Advunc,c,.\ in I./(,c,lrophorc,.\/.\ (C’hrambach, A,, Dunn, M. & Radola. B. J., eds.), vol. I. VCH Verlagsgesellschaft, Weinheim, FRG, in the press
Gershoni, J. M. (1988) in Methods oJ’Hioc~hemiculAnulysis (Glick,
D., ed.), vol. 33, John Wiley, New York, in the pre\\
Mosckovitz, R. & Gershoni, J. M. ( 1988)J . H i o l . <’hem.in the press
Neumann, D., Gershoni, J. M., Fridkin, M. & Fuchs. S. ( 1985) I’roc.
Narl. Acad. Sci. U.S.A.82. 3490-3493
Neumann, D., Barchan, D., Safran, A,, Gershoni, J. M. & Fuchs, S.
(1986) Proc. NU^. Acud. Sci. U.S.A.83. 3008-30 1 1
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Towbin, H . & Gordon. J. ( 1984) J . Immunol. Meihod.v 72. 3 13-340
Wilson, P. T., Gershoni, J. M., Hawrot, E. & Lentz, T. L. (1984)
I’roc. Natl. Acud. Sci. U.S.A. 81, 2 5 5 3 - 2 5 5 7
Received 3 November 1987
Non-radioactive probes in Southern blots
ALAN D. B. MALCOLM and HERMIA FIGUEIREDO
l)epurttnerit of’Riochernistty, C’haritig C’ross arid Westmitister
Medicd School, Fiilhuni I’uluce Roud. Lotidori Wh 8RFs U.K .
The use o f DNA probes as diagnostic reagents is becoming
increasingly common [ 1 I. Thus they are able to diagnose with
great precision and sensitivity infectious (e.g. viruses) (2, 31,
inherited (e.g. sickle cell disease) 14, 51 and acquired (e.g.
somatic cell changes leading to tumours) diseases [6]. DNA
probes have several advantages over the more conventional
biochemical techniques for the investigation of disease. Thus
the rules by which a DNA probe recognizes its complementary partner have been well understood since the days of
Watson and Crick. This is in complete contrast to the rules
governing the interaction between an enzyme and a substrate
or between an antibody and an antigen. In one or two individual cases we have a good understanding, but no general set
of rules is yet available. A consequence of this is that it is
possible both theoretically and in practice to design a probe
specific for any particular purpose [ 71.
A further advantage which nucleic acids have is that all
nucleic acid molecules are chemically and physically very
similar to each other in being long repeating negatively
charged polymers. This means that they all display similar
behaviour in response t o physical and chemical changes, and
makes dealing with them much more predictable (and hence
reproducible) than dealing with, say, enzymes or antibodies.
The third advantage which nucleic acids have is that it is
possible to detect a particular genome (abnormal o r normal)
even in the absence of the gene product for which it codes.
Again this is in contrast to the use of enzymic methods or
Vol. 16
antibody methods where transcription and translation must
both have taken place. Thus sickle cell disease can be
diagnosed it1 liter0 [ 8 ] and latent viruses can be diagnosed in
tissues before the disease has taken hold 191. Even more
remarkably, DNA probes can be used which are not even
causally linked to the disease at all. Thus restriction fragment
length polymorphisms can be detected where the base
change(s) which give rise to the restriction fragment length
polymorphism is genetically linked t o the locus causing the
disease but does not itself give rise to it [ 101.
The tremendous power of DNA probe technology has not
surprisingly, therefore, given rise to a wide range of technologies to enable these advantages to be exploited. Southern
blotting [ 1 11 allows the transfer of DNA fragments from a gel
after electrophoresis on to a suitable membrane such as
cellulose nitrate or modified nylon. Spot blots and slot blots
simply employ a manifold to allow a sample to be spotted on
to the membrane which can then be immobilized by baking
[12]. In all these cases, the sample is immobilized to a
membrane and after denaturation is then hybridized t o a
suitable probe.
A further advantage which nucleic acids have over other
approaches is the wide range of methods available to
produce a labelled probe. Enzymes exist which allow us to
perform nick translation [ 131, random oligonucleotide
priming [ 141, polynucleotide kinase labelling [ 1.5I and the
addition of tails using terminal deoxynucleotidyltransferase
[ 161; reverse transcriptase allows us to make a labelled DNA
copy from an RNA template [ 171. The chemistry of oligonucleotide synthesis plus chemical methods for labelling
DNA can all be added to this list [18]. A wide variety of