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Introduction_________________________________________________________________________________
____
The idea that protein-based cell-surface receptors mediate biological signalling and
downstream cellular processes is the accepted theory to explain tissue responses to
pharmacological agents and endogenous signal molecules. The theory was initially proposed
by British pharmacologist J.N. Langley. His seminal paper proposed that all cells possess a
chief substance and a receptive substance with the latter possessing the potential to
affect the metabolism of the former1. The theory of cellular receptors was extremely
controversial, with scientists sceptical of their existence as anything more than abstract
concepts. It was in this environment that Robert Lefkowitz began his research on receptor
mediated signalling. During his 40 years of research, Lefkowitz revolutionized biochemistry
and cell biology, providing near undeniable proof for the existence of cell-surface receptors
and their roles in homeostasis and the cell response.
In particular, Lefkowitz dedicated his research to seven transmembrane spanning, or G-protein
coupled receptors (GPCRs). This enormous receptor family comprises six different classes, and
GPCRs are encoded by roughly 800 human genes (~4% of the genome) 2. GPCRs are a target
for roughly 40% of all modern pharmaceuticals, and research on these receptors is still crucial
to modern therapeutics. Lefkowitz was an early pioneer in the radiolabeling studies that led to
isolation of the alpha and beta adrenergic receptors, two GPCRs involved in nervous system
response. This work, alongside his -arrestin studies contributed to his winning the 2012 Nobel
Prize in chemistry alongside colleague Brian Kobilka. Currently, the Lefkowitz lab continues to
work on GPCRs and their downstream effectors through a variety of modern lab techniques
such as bioluminescence resonance energy transfer and the use of biased ligands.
Early Studies and the Beginning of Lefkowitzs
Work____________________________________________________
One of the first experimental suggestions that receptors exist and may be classified by their
downstream cellular response was by Raymond Ahlquist in 1948. Ahlquists experiment
showed that animals exhibited two distinct physiological responses when treated with various
amine compounds. The responses were seen as generally excitatory or inhibitory. This
observation was rationalized by proposing
two distinct receptors, which Ahlquist named
alpha and beta3. This experiment formed the
basis of Lefkowitzs research.
Lefkowitz began his research on the adrenergic
receptor
(-AR)
through
radiolabeled ligand studies. The research
group used tritiated (-)alprenolol as a antagonist
to
study
ligand-receptor
interactions4. Frog erythrocytes membranes
incubated with
(-)
3
[ H]alprenolol for various timespans were
observed via scintillation spectroscopy,
which measures gamma-ray emission, to
determine the extent of ligand binding. The
results of the experiment showed that
alprenolol bound rapidly and reversibly to the
proposed -adrenergic receptor. The addition
Figure 1: The time-dependant binding of
of stronger agonists such as
(-)propranolol
tritiated alprenolol to frog erythrocyte
caused quick dissociation of alprenolol
membranes
at
various
temperatures.
(Figure 1). In conjunction with the binding
assay, the group carried out adenylate
Alprenolol
showed
rapid
binding
to
cyclase activity assays. Membrane fragments
membrane fragments; this was readily
were
incubated
with
the
-agonist reversed by addition of the stronger
isoproterenol and [32P]ATP. Cyclase activity
was followed via scintilliation spectroscopy after isolating the 32P labeled cAMP. The assay
showed a marked increase in adenylate cyclase activity from basal levels following the
addition of isoproterenol, but activity was inhibited with the addition of propranolol (Figure 2).
This inhibition was rationalized as the antagonist propranolol displacing alprenolol at the
receptor binding site, stalling signal transduction. This experiment not only fortified the
argument for receptor coupling to secondary messengers such as cAMP, but also provided a
novel approach to identifying receptors, binding sites, and corresponding ligands. This
experimental procedure was used in subsequent studies to determine binding sites for the adrenergic receptor5. Rabbit uterus cells were incubated with a tritiated antagonist. The
experiment once again showed ligand binding to the
membrane. Addition of the -antagonist propranolol did
not displace the -antagonist, indicating non-redundancy
of the ligand binding sites.
Following initial characterization of the alpha and beta receptors, the Lefkowitz lab began to
study a phenomenon associated with agonist binding. Competition curves produced from
radiolabeled ligand binding on frog erythrocyte -receptors showed a variation from agonist
and antagonist binding. Antagonist curves showed a slope-factor of ~1, whereas agonist
curves showed a slope-factor of ~0.83. This variation, present in a variety of receptor classes
including the muscarinic and glucagon receptors, needed to be rationalized with more
complex model than simple agonist-receptor association. The group proposed several binding
models and computationally fit them to the experimental competition curve of the -agonist
[3H]hydroxybenzylisoproterenol (HBI)6. They determined the most probable model to be
ternary-complex formation (Figure 3). The model describes agonist and receptor forming a
ternary complex with an unknown protein X. This ternary complex corresponded with a highaffinity state; dissociation of X from the agonist-receptor complex produced the low affinity
state where ligand readily dissociates. The proportion of high and low affinity receptors was
perturbed by the addition of GPP(NH)P, a non-hydrolysable GTP analogue. This nucleotide
regulatory effect was also observed in turkey erythrocytes. Based on the ternary-complex
model, and on earlier experiments implying adenylate
cyclase could be decoupled from receptor action 7, Figure 2: Adenylate cyclase
Lefkowitz theorized that a final effector was activated by
activity
assay
of
frog
the receptor following GTP stimulation. The GTP
erythrocyte
membranes
regulatory effect immediately implicated the recently
stimulated
with
varying
discovered GTP-binding regulatory protein as the
concentrations
of
unknown effector. Building upon previous studies which
isoprotenerol.
Isoprotenerol
showed an apparent increase in receptor size upon
8
activates adenylate cyclase in
agonist binding , the Lefkowitz lab aimed to study the
role of G-protein binding in adrenergic receptor action.
a dose dependant fashion.
Gel elution experiments showed that
rat reticulocyte adrenergic receptors
eluted as larger complexes when
agonists bound (Figure 4), this
process quickly reversed following
the addition of GPP(NH)P. Cholera
toxin was then used to ribosylate
reticulocyte
G-proteins
with
[32P]NAD. Elution of ligand-bound
receptor complexes showed elution
volumes
characteristic
of
the
ribosylated
G-protein.
These
experiments directly demonstrated
the dual role of ligand and effector in
the regulation of receptor activity. At
Figure 3: A schematic diagram of the ternary complex
this point Lefkowitz proposed a
mechanism of receptor action which
model proposed by Lefkowitz et al. In the model, the
formed the basis of current theories
receptor (R) binds both agonist (H) and the unknown
of GPCRs. The model describes
effector (X) to form a high affinity complex. Addition of
ligand and G-protein binding to the
GTP drives activation of the final effector (E),
receptor, forming the high affinity
complex. GTP binds to the G-protein, dissociation of X, and formation of the low affinity
causing G-protein dissociation and formation of the low affinity ligand-receptor complex. The
active G-protein then regulates activity of adenylate cyclase until intrinsic GTP hydrolysis
deactivates the enzyme.
terminus. Treatment of the degraded receptor with endoglycosidase F truncated the receptor
further, indicating glycosylation of both N-terminal Asn residues. The experiment showed the
topology of the receptor helices, and how the glycosylated ligand binding site is located Nterminally to the effector binding sites.
Upon topological characterisation, the group carried out site directed mutagenesis studies on
the cytoplasmic loops of the human -adrenergic receptor19. The group once again inserted
these proteins into X. laevis oocytes using more modern mRNA transfection techniques.
Solubilized cell membrane fragments of the oocytes were then assayed for adenylate cyclase
activity following isoproterenol stimulation. Mutations in the third cytoplasmic loop and the
cytoplasmic tail caused a stark decrease in adenylate cyclase activity, while mutations in the
second cytoplasmic loop showed a modest decrease in activity. From this, the group proposed
that the cytoplasmic tail and cytoplasmic loop 3 played the largest role in G-protein binding,
whereas a conserved cysteine residue in C-loop 2 played a more obscure role in G-protein
orientation. Further proof of the role cytoplasmic loops play in G-protein binding was
discovered through the production of chimera receptors by Brian Kobilka in the Lefkowitz lab.
Mutagenesis of the third cytoplamsmic loop of the 2 receptor to a 2 sequence was enough
to change the receptor response from inhibitory (Gi coupled) to stimulatory (Gs coupled) with
respect to adenylate cyclase activity 20.
By the end of the 1980s the Lefkowitz lab had laid the foundation for modern understanding
of the biochemical and physiological role of GPCRs. The labs work was built upon by multiple
researchers across the globe, most importantly Brian Kobilka and his crystal structure
research carried out at Stanford.
Receptor
Desensitization,
Discovery
of
-Arrestins,
and
Current
Work____________________________________
In addition to Lefkowitzs work on the structural characterization of GPCRs, he carried out
early research into the phenomenon of receptor desensitization. Lefkowitzs early studies on
turkey erythrocytes showed phosphorylation of -adrenergic receptors following long term
agonist exposure. This phosphorylation coincided with decreased stimulation of adenylyl
cyclase, indicating a potential mechanism for desensitization and signal quenching 21. To
determine the kinase responsible for -AR
phosphorylation,
the
group
purified
hamster lung -AR and incubated the
receptor with kin- cell lysates, cells which
exhibit receptor desensitization, but did not
possess cAMP dependant kinases. The
receptor showed phosphorylation in an SDS
gel. The kinase was then isolated through
HPLC, and initially named -adrenergic
receptor kinase (ARK)22. However, a
perplexing phenomenon occurred, whereby
highly purified ARK lost its ability to
desensitize the -AR, implying the activity
of a second unknown protein. Concurrent
studies by Kuhn et al. showed the activity
of a protein, named arrestin, in the
desensitization pathway of rhodopsin. The
Figure 6: The GTPase activity of -AR in the
Lefkowitz group obtained arrestin from the
presence of arrestin (o), -arrestin1 (), and Kuhn laboratory and was able to show
arrestin2 (). Both -arrestins show potent and
restoration
of
ARK
desensitization
23
following addition of arrestin . Though specific inhibitory effects, whereas arrestin
arrestin mediated desensitization, the
shows a more moderate inhibition.
protein isolated to the retina and could not be the physiological agent for -AR
desensitization. Knowing this, Lefkowitzs lab scanned bovine and rat brain cDNA libraries,
revealing several proteins with >70% sequence homology to arrestin. These proteins showed
highly specific and potent desensitization of the -adrenergic receptor and were named arrestins (Figure 6) 24 25.
2.
3.
Ahlquist, R. (1948) A study of the adrenotropic receptors. Am. J. Physiol. 153, 586600.
4.
5.
6.
DeLean, A., Stadel, J. M. & Lefkowitz, R. J. (1980)A ternary complex model explains the
agonist-specific binding properties of the adenylate cyclase-coupled beta-adrenergic
receptor. J. Biol. Chem. 255, 710817.
7.
Stadel, J., DeLean, A. & Lefkowitz, R.J. (1980) A high affinity Agonist beta-adrenergic
receptor complex is an intermediate for catecholamine stimulation of adenylate cyclase
in turkey and frog erythrocyte membranes. J. Biol. Chem. 255, 14361441.
8.
9.
10.
11.
12.
Benovic, J. L., Shorr, R. G., Caron, M. G. & Lefkowitz, R. J. (1984) The mammalian beta 2adrenergic receptor: purification and characterization. Biochemistry 23, 45108.
13.
Regan, J. W., Nakata, H., DeMarinis, R. M., Caron, M. G. & Lefkowitz, R. J. (1986)
Purification and Characterization of the Human Platelet 2-Adrenergic Receptor. J. Biol.
Chem. 261, 38943900.
14.
Dixon R.A. et al. (1986) Cloning of the gene and cDNA for mammalian beta-adrenergic
receptor and homology with rhodopsin. Nature 321, 7579.
15.
Engelman, D. M., Goldman, A., & Steitz T.A. (1982) The identification of helical segments
in the polypeptide chain of bacteriorhodopsin. Methods in enzymology 88, 81.
16.
Frielle, T. et al. (1987) Cloning of the cDNA for the human beta 1-adrenergic receptor.
Proc. Natl. Acad. Sci. U. S. A. 84, 79204.
17.
Kobilka, B. K. et al. Cloning, sequencing, and expression of the gene coding for the
human platelet alpha 2-adrenergic receptor. Science 238, 6506 (1987).
18.
Dohlman, H. G., Bouvier, M., Benovic, J. L., Caron, M. G. & Lefkowitz, R.J. (1987) The
Multiple Membrane Spanning Topography of the B2-Adrenergic Receptor. J. Biol. Chem.
262, 1428214288.
19.
20.
21.
22.
23.
24.
Lohse, M., Benovic, J. & Codina, J. Caron, M.J., & Lefkowitz R.J. (1990) beta-Arrestin: a
protein that regulates beta-adrenergic receptor function. Science. 248, 15471550.
25.
26.
Lin F.T., Daaka Y., Lefkowitz R.J. (1998) beta -Arrestins Regulate Mitogenic Signaling and
Clathrin-mediated Endocytosis of the Insulin-like Growth Factor I Receptor. J. Biol. Chem.
273, 3164031643.
27.
Fisher, A et al. (1993) Selective signaling via unique M1 muscarinic agonists. Ann. N. Y.
Acad. Sci. 695, 3003.
28.
29.
Shukla, A.K., Violin, J.D., Whalen, E.J., Gesty-Palmer, D., Shenoy, S.K., & Lefkowitz, R.J.
(2008) Distinct Conformational Changes in Beta-Arrestin Report Biased Agonism at
Seven-Transmembrane Receptors. Pro. Natl. Acad. Sci. U.S.A. 105, 998893.