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Robert Lefkowitz: The Discovery and Characterization of Gprotein Coupled Receptors (GPCRs)

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.

Receptor Purification and Characterization_______


With a basic model for GPCR action, the Lefkowitz lab began the task of isolating adrenergic
receptors for further characterization. To isolate the receptors, the group developed affinity
chromatography columns with immobilized alpha and beta antagonists. Solubilized

erythrocyte membrane fragments were


eluted through the columns, allowing
purification of all four isoforms of the
adrenergic receptors 1, 2, 1, and 2.
SDS gels showed the proteins as single
polypeptides roughly 64kDa in size9. To
investigate
whether
these
isolated
receptors
conveyed
response
to
adrenaline Lefkowitzs lab reconstituted
the receptors in lipid vesicles. These
vesicles were fused with Xenopus laevis
erythrocytes,
which
possessed
prostaglandin sensitive adenylate cyclase
but little -AR activity10. The hybrid cells
showed
a
2-5
fold
increase
in
responsiveness to adrenaline, indicated
by an increase in [32P]cAMP concentration
(Figure 5)11. Using the same procedure,
characterization of mammalian 2 and 1
receptor activity followed 1213.

Figure 4: The elution volumes of agonist


bound () and antagonist bound () adrenergic receptors. Agonist binding shows a
decrease in elution volume, indicating an
apparent increase in receptor size. Elution
volume was determined through scintillation
spectroscopy of the bound ligand.

Following receptor isolation, Lefkowitzs


group partnered with the MSD laboratories
to sequence the -AR gene14. Hamster
lung -AR was digested using cyanogen
bromide, which cleaves peptide bonds cterminally to methionine. The resulting
oligopeptides were sequenced using Nterminal sequencing and complimentary
radiolabeled
oligonucleotides
were
synthesized to isolate the -AR DNA from a
bacteriophage library. Once isolated and
sequenced it was immediately noticed that
the -AR gene shared strong sequence
homology with other known receptors. The
-AR showed several conserved sequences
homologous to the light sensing receptors
rhodopsin and bacteriorhodopsin this
indicated similarity in function between the
two receptors types. The structure of
Figure 5: The fold increase of X. laevis
rhodopsin was shown in previous studies to
erythrocyte
adenylate
cyclase
activity
possess seven transmembrane spanning 15
following
reconstitution
with
-AR
containing
helices . With these new experimental
lipid
vesicles
and
stimulation
with
results, Lefkowitz and his colleagues began
to unveil the ubiquitous nature of GPCR
isoprotenerol. (BAR = -AR)
structure, and the potential for receptor stimulation through non-ligand interactions. Within a
few years of cloning the -AR gene, labs across the world began to characterize multiple
receptor subtypes, including the human 2 and 1 receptors16 17
With the success of cloning receptor genes, the Lefkowitz lab carried out experiments aimed
at understanding the structural basis of ligand binding and G-protein coupling in GPCRs 18. To
determine the topology of the receptors, radiolabeled -AR in lipid vesicles and in intact cells
were trypsinized. The proteins were then purified and run on SDS-gels to determine the sites
of cleavage. Both experiments showed a single cleavage site, producing two fragments of size
38 and 26 kDa. The fragments corresponded to cleavage at amino acid 180. As trypsin was
only able to cleave from the extracellular side, it was proposed that the loop containing
residue 180 resided on the extracellular surface between transmembrane helices four and
five. The group then treated reconstituted receptors with the enzymes carboxypeptidase Y
and endoglycosidase F. Treatment with carboxypeptidase Y produced truncated receptors
degraded at the C-terminus up to the transmembrane helix. Degradation removed two
potential N-glycosylation sites, leaving two Asn residues for glycoslylation near the N-

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.

Building upon works at other research labs


which suggested that arrestins mediate
receptor endocytosis, the Lefkowitz lab began
to study the mechanism of -arrestin
desensitization.
Lefkowitz
forced
overexpression of the IGF-1 receptor, a
receptor tyrosine kinase, in HEK 293 cells. The
cells
were
transfected
with
-arrestin
expressing vectors, and stimulated with the
agonist IGF-1. Using flow cytometry with
immunoflourescent receptor tags, the group
showed a substantial increase in receptor
internalization in cells overexpressing arrestin compared to control (Figure 10)26. In
addition to these experiments, the group
characterized the effect that -arrestin plays in
novel divergent signalling pathways. The group fed overexpressing cells tritiated thymidine
and followed total DNA synthesis via scintillation spectroscopy. The experiment showed a
nearly two-fold increase in DNA synthesis for cells overexpressing -arrestin, alongside a
marked increase in phosphorylation of downstream effectors such as MAP-kinase. These
experiments showed that -arrestins not only work to desensitize receptor signals, but also
mediate downstream signalling processes of their own.
This dual function of receptors to mediate signalling through effectors such as G-proteins and
-arrestins forms the basis of Robert Lefkowitzs current work. Currently, the Lefkowitz lab
works on the characterization of receptor responses stimulated by biased agonists. A biased
agonist is a ligand which triggers receptors to undertake certain conformations biased to the
recruitment of particular effectors at the expense of others. The idea of biased agonists was
proposed from studies on acetylcholine receptors. Upon agonist stimulation the receptors are
known to stimulate Gs coupled cAMP production and Gq coupled phospholipase C activation.
However, later studies showed that some agonists may stimulate one pathway while inhibiting
another 27.
Figure 7: The percent agonist promoted
With this experimental basis, the Lefkowitz
IGF1 receptor internalization in HEK 293
group has begun studying the structural and
cells containing vectors containing various
mechanistic basis of ligand bias in GPCR
-arrestins and their serine-412 mutants
signalling. Using modern techniques such as
mass spectrometry and proteomics, the group
(S412A/S412D). Cells overexpressing most
produced phosphorylation maps of HEK 293
isoforms of -arrestin showed increased
cell lysates stimulated with a -arrestin biased
receptor
internalization
compared
to
agonist of the angiotensin receptor. The
control
(mock).
Analogous
experiments
on
mapping revealed the role of -arrestin in
GPCRs showed similar results.
stimulating multiple G-protein independent
signals such as the MAP kinase, and PI3K pathways 28. In addition to this mechanistic work, the
lab uses techniques such as bioluminescence resonance energy transfer to study
conformational changes in receptor and effector upon biased ligand binding. Lefkowitz and
Shukla29 have shown the biased ligands induce conformational changes in intracellular
effectors such as -arrestin, indicating a structural basis for biased ligand stimulation. The
current research at the Lefkowitz lab is aimed at utilizing knowledge of ligand bias in the
development of novel therapeutics which selectively target G-protein and -arrestin pathways.
These types of novel therapeutic are potential replacements for current drugs targeting
pharmacologically important receptors such as angiotensin and -opoid receptors.
References:_______________________________________________________________________________
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