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evidence had been developed which indicated that the multiple phosphor-
ylation of the
b
2AR which accompanied so-called homologous- or
“agonist-specific” desensitization of the
b
2AR was carried out by a novel
cAMP-independent kinase, initially named the
b
-adrenergic receptor kinase
or
b
ARK.
16
The discovery of this enzyme in 1986 was part of a rapidly mov-
ing story which would, within the space of just a few years, firmly establish a
very general paradigm for a mechanism of desensitization of G protein-
coupled receptor signaling.
Jeff Benovic, then a very talented graduate student in my lab, was busy
purifying
b
ARK from bovine brain during this period.
17
To assay its cata-
lytic activity through various stages of purification, he measured its ability to
phosphorylate
b
2AR (purified from endogenous sources, no cloned or
overexpressed material was available during this era) reconstituted in phos-
phatidylcholine vesicles. He could then assess the “uncoupling” or desensi-
tization of the receptor by following its ability to mediate stimulation of the
GTPase activity of co-reconstituted G
s
. Paradoxically, the more he purified
the enzyme and enhanced its specific catalytic kinase activity, the lower
became its “desensitizing” ability.
18
We feared we were losing some neces-
sary cofactor or accessory component. However, a variety of biochemical
approaches failed to yield the putative missing element.
While we pondered this situation in 1986, Kuhn published a report that
48K protein binds to photoexcited rhodopsin and quenches phosphodiester-
ase activation.
6
This report really caught our attention, since the 48K protein
(arrestin) seemed to match the profile of our missing desensitizing element.
However, we knew what we were looking for could not be arrestin itself
since the expression of arrestin was known to be limited to the retina and
pineal gland. Undeterred, I called Kuhn and persuaded him to send me some
of his arrestin protein. Benovic was then able to demonstrate that when
added to his reconstituted system (albeit at high arrestin/
G
s
molar ratios),
it restored the desensitizing ability of the purified
b
ARK.
18
This led us to
the hypothesis that what we were purifying away from
b
ARK might be a
protein analogous to and possibly homologous with visual arrestin.
When, in 1987, Shinohara cloned “48K” protein,
19
we obtained his
clone and Martin Lohse in our lab was able to use it to clone a 70% sequence
identical protein we named
b
-arrestin.
20
Not long thereafter, another fellow
in the lab, H˚vard Attramadal cloned another member of this family and we
named it
b
-arrestin2, now referring to
b
-arrestin as
b
-arrestin1.
21
In contrast
to visual arrestin,
b
arr1 and 2 were found to have much higher affinity for
phosphorylated
b
2AR than for phosphorylated rhodopsin.
21
Moreover,
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