10 0 --O.. __• __... E . ..... ............. •
C ., I:! KH - 24 nM , "0 , >- KL -1400nM s: . \
Ci 20 °/.RH 77 °/. ., 'I' °/. RL .23°/. ....
( - ) Isoproterenol concentration (-109lo Molar)
(GTP) in frogerythrocyte membranes. Ligand concentration was - 1.5 nM. (Kent erat., 1980).
radiolabelIed antagonist [JH]-dih ydroalprenolol ([JH]D HA ). The curves are show n
in th e presence and ab sen ce of the gua ni ne nucleotide GTP which is required for
hormone stim ulation of adenylate cycla se. The curves shown are theoretical
computer generated best frts for the experimental data (Kent, DeLean & Lefkowitz ,
1980) . The antagonist curve shown is typical of all antagonist curves in this system
and is weil frtted by a sim ple Michaelian 'steep' (slope factor = I) curve drawn simply
according to the law of ma ss ac tion applied to the competition of a competitor and
the radioligand for a single class of noninteracting receptor sites (Kent er. al.. 1980).
The curve is un affected by the presence ofguanine nucleotide.
T he curves for all agonists suc h as IP are markedly different. These curves are
'sha llow' (slope factor « 1), cornplex, multiphasic, and are shifted to the
right and steepened to a slope factor of I by guanine nucleotides (Kent er al.. 1980;
Lefkowitz, Mullikin & Caron , 1976). Computer modelling of these curves indicates
that they are best frtted by a model for two classes ofsites wh ich, though they bind the
antagonist radioligand with equal affmity, bi nd the competing agonist with two
distinct affmities, one being considerably higher than the ot her (Kent er al.. 1980). In
the particular example shown, 75% ofthe rece ptors are bi nding the ago nist with high
affmity and abo ut one-quarter of the receptors are binding the agonist with lower
(60-70 fold lower) affmity, In the presence of guanine nucleotide, however, all ofthe
receptors are now found in the low affinity state. The unique affm ity of agonist
binding observed in the pre sence ofguanine nucleotide appears to match the lower of
the two affmities for agonist binding observed in the absence of guanine nucleotide
(Kent er al., 1980). Also, the actual number of ß-adrenergic receptors in the
membranes is not changed by guanine nucleotides. These data suggest that guanine
nucleotides mediate a transition of high to low affinity agonist binding sites. T hese
and other data which will not be reviewed here ha ve suggested that the un ique
property wh ich distinguishes agonists from antagonists and which is apparent in
binding studi es is the ab ilit y of agonists to distinguish between high and low affmity
DESENSITIZATI ON AN Dß -ADRENERG IC RECEPTOR FUNCTION 149
states ofthe p-adrenoceptor (Kent et al.,1980). Stud ies with a wide variety ofpartial
agon ists with varying intrinsic activities indicate that the ability to induce or stabilize
th is high affmity form ofthe receptor correlates directly with the ability ofthe drug to
stimulate adenylate cyclase activity.
Recentl y other data have suggested that the high affmity guan ine nucleotide
sensitive form of the p -adrenergic receptor induced by agonists is, in fact, a ternary
complex of agonist, receptor, and guan ine nucleot ide regulator y protein (Limbird &
Lefkow itz, 1978; Limbird, Gill & Lefkowitz, 1980; Limbird , Hickey & Lefkow itz,
1979; Limbird, G ill, Stadel , Hickey & Lefkowi tz, 1980; DeLean , Stadel & Lefkowitz,
1980). Agonists of increasing intrinsic activ ity show progressively greater ab ility to
promote the combina tion ofthe hormone receptor complex (Iow affmity form) with
the nucl eotid e regulator y protein to form th is tern ar y complex (De Lean et al., 1980) .
Th e tern ar y co mplex of agonist, receptor and nucl eotid e site (HRN) appea rs to be a
tran sient rcaction intermediate, the format ion of which is required for the activa tio n
of adeny late cyclase (Stadel. DeLean & Lefkowitz, 1980). A schema which
summarizes these interactio ns is shown below:
X=some other membrane component, presumably the guan ine
nucle ot ide regulatory protein.
DeLean et al. (1980) have shown, using computeriz ed curve fitting pro cedures,
that th is model closely accomodates all our bind ing data in the p-adrenergic
receptor system of the frog eryt hrocyte and that it applies as weil to several other
p-adrenergic receptor systems. The computer modelling, of course, cannot reveal
the identity of the extra compo nent involved in the ternar y complex model.
However, independent biochem ical evidence suggests that it is, in fact, the nucleotide
regulatory site (Limbird et al., 1978, 1979, 1980a , 1980b ).
Ligand bind ing studies with the rad io-Iabelled p -adrenergic receptor agonist
[JH)-h ydro xybenz ylisop roterenol ([JH)HBI) have also been performed (DeLean et al.,
1980; Lefkow itz & Willi am s, 1977; William s & Kefko witz, 1977 ). Thi s is a full
agoni st with high potency in p l adrenergic receptor systems. Binding studies with
thi s radiolabelIed agonist serve to label onl y the highe r ofthe two affmity states ofthe
p-adrenergic receptor and thu s provide a convenient way of assessing only the high
affmity, 'coupled' guanine nucl eot ide-sensitive form ofthe p-adrenergic receptor.
G iven these methods, not only the number of p -ad renergic receptors but the
status of their coupling with the presum ed nucleotide regulator y binding site after
desensitization can be examined. An example of such data is shown in Figure 3a
(Kent et al., 1980). Shown are competition curves ofthe agonist IP with [JH)DHA in
membranes der ived from control and IP desensitiz ed cells. The computer modelIed
param eters are shown in the figure. It is obviou s that there are severa1alterations in
the receptors as revealed by the se studies. First of all, there is a very obvious,
approximately 70% fall in receptor number. Thus, there is mu ch less DH A binding
in membran es from the desensitized cells. However, as shown by the computer
modelling there are also significant alteratio ns in the ab ility of the
agonist to indu ce the high affmity coupled form ofthe rece pto r. T hus, the affinity of
(-) Isoproterenol concentratlon (-lo9lo Molar)
Figure 3a Computeri zed curve fitting of binding data from displacement of (-)PH]-
dihydroalprenolol by (-)isoproterenol (isoprenaline) in control membranes and membranes
derived from desensitized cells. Whole frog erythrocytes were desensitized by incubation ofthe
cells with 0.1 mM isoproterenol for 3 h. The total decrease in maximal (-)[3H]-
dihydroalprenolol binding was 72%. KLiKH=113 in control membranes and 44 in membranes
from desensitized cells in this experiment. (Kent et al., 1980).
the high a tTmity form is several fold lower and the percentage ofreceptors in the high
affmity state is also significantly decreased . The da ta in Figure 3b indicate the results
of five experiments in which it was shown that th ere is a statistically signiftcantly
(P < 0.05) impaired ability ofagonists to form the high affmity state ofthe receptors
in membranes from desensitized cells. These data strongly suggest so me impairment
in the coupling ofthe receptor to the nucleotide protein which is normally promoted
Similar conclusions can be reached by direct binding studies with the radiolabelIed
agon ist [3H]HBI (WesseIls , Mullikin & Lefkowitz , 1978; Wes sels, Mullikin &
Lefkowitz , 1979). In th ese studies it was found that, on a percentage ba sis, there was a
signi fica ntly great er fall in [3 H]H BI binding than in [3H]DHA binding to membranes
after desen sitization. This is because, as noted above, [3H]HBI lab els on ly the high
atTmity 'coupled' form of the receptor whereas [3H]DHA labels the entire receptor
population. Since both receptor number and high affmity receptor state formation
are decreased, this leads to a greater loss of [3H]HBI than[JH]DHA binding. These
data suggest then that the high affmity intermediate (presumably HRN) is an
that interventions wh ich impair the ability to form the high affmity state of the
receptor also decrease the ab ility of agon ists to desen sit ize receptors as assessed by
the ability to promote receptor loss (Wessels eral., 1979 However , other studies show
that there are conditions under whi ch the ability to form the high affm ity agonist
receptor complex is retained and yet one is still not abl e to promote receptor loss
(Stadel & Lefkowitz, 1979).These fmdings suggest that although formation ofthe high
affmity receptor intermediate is necessary for desen sitization, its formation is still not
sufficient. The nature ofother processes which ma y be in vol ved remains unknown.
That th e receptors are, in fact, the major locus of a lterations in homologous
desensitization in these cells has been furt her supported by data recently obtained by
DESENSITIZATION AND ß-ADRENERGIC RECEPTOR FUNCTION 151
Pike & Lefkowitz (1980). Cell fusion techniques, originally described by Orly &
Schramm (1976), were used in which the receptors from one cell can be coupled
Figure3b ElTect of desensitization on high and low affmity state receptor concentrations.
Concentrationsofhigh (RH) and low(RL) alTmity state receptorsweredetermined by computer
modelling. Resultsare expressed as mean ± s.e.mean for liveexperiments. Afterdesensitization
the proportionsofhigh affmityreceptorsdecreased significantly (P < 0.05). Kent et al.. 1980).
to cyclase components from other cells. Cells were desensitized and then used as
either the receptor (R) or 'cyclase' donors for such fusion studies. The data indicated
that when receptors are derived from desensitized cells , the resulting hybrid showed
desensitization. By contrast, ifthe more distal components ofthe system are derived
from desensitized cells, no desensitization is seen in the hybrid. These data further
support the notion that it is the receptors, in terms of both their number and their
ability to become coupled to distal components such as the nucleotide sites which
are altered in homologous desensitization as expressed in the frog erythrocyte. Other
recent data (Su, Harden & Perkins, 1979, 1980) is compatible with these not ions and
suggests further interesting information about the sequence of events. Studying an
astrocytoma cell in culture, this group has found that homologous desensitization by
catecholamines is associated with the same two uncoupling and receptor loss lesions
described above. However, in contrast to the frog erythrocyte, where both processes
appear to proceed with fairly similar time courses, in astrocytoma cells the
uncoupling lesion, that is, the lesion manifest by the inability to form the high affmity
state of the receptor appears to temporally precede receptor loss. This group (Su et
al., 1979,1980) has suggested that the uncoupled form ofthe receptor may represent
an earlier stage in the desensitization process and that the uncoupled receptors are
then eventually lost, perhaps by internalization. It is possible that, depending on the
kinetics ofthe formation ofthe uncoupled receptors and the 'Ioss' ofreceptors that, at
any given point in time, in a particular system, one might see varying combinations
of receptor uncoupling and receptor loss. Thus, if receptor loss occurs rapidly after
uncoupling the two processes would appear to occur in parallel (for example, frog
erythrocyte). By contrast, if receptor loss was slow there would be a build-up of
uncoupled receptors as is the case in the astrocytoma cel!.
In summary, recent radioligand binding studies of ß-adrenergic receptors are
shedding new light on the mechanisms by which agonists Iead to desensitization. Our
studies suggest multiple alterations in the receptor component of the system. These
alterations lead both to I) an impaired ability ofthe receptors to form the high affmity
coupled state which leads to adenylate cyclase activation as weil as 2) an actual loss
in the number of functional receptors in the membranes. These studies do not
exclude the possibility of more distal alterations in the system contributing to the
desensitization process. However, they do suggest that in homologous desensitization
the receptor may be the major locus of such alterations. In contrast, a variety of
studies in systems showing heterologous desensitization (not reviewed here) suggest
that the receptors themselves may be relatively unperturbed but that alterations at
more distalloci such as the nucleotide regulatory site, for exarnple, may be relatively
more important. Receptor binding studies provide an important and powerful new
tool for assessing such alterations. However, their optimal use requires more than
simple enumeration ofreceptor number, but also detailed studies of agonist-receptor
Chuang, O. M. & Costa, E. (1979). Evidence for intemalization of the recognition site of
ß-adrenergic receptors during receptor subsensitivity induced by (-)isoproterenol. Proc.
Nat . Acad. Sei. USA . 76, 3024-3028.
Oe Lean, A., Stadel, J. M. & Lelkowitz, R. J. (1980). A temary complex model explains the
agonist-specific binding properties ofthe adenylate-cyclase coupled ß-adrenergic receptor.
J. biol. Chem .. 255, 7108-7117.
Harden , T. K., Cotton, C. U., Waldo, G. L., Lutton ,J. K. & Perkins,J. P. (1980). Catecholamine
induced alteration in the sedimentation behavior of membrane bound ß -adrenergic
receptors . Science. in press.
Kent, R. S., Oe Lean, A. & Lelkowitz, R. J. (1980). Quantitative analysis of ß-adrenergic
receptor interactions: resolution ofhigh and low affinity states ofthe receptor b y computer
modellingofligand bindingdata. Mol. Pharrnac.. 17, 14-23 .
Lelkowitz, R. J., Mullikin , O. & Caron , M. G. (1976). Regulation of ß-adrenergic receptors by
S'guan ylyl-imidodiphosphate and other purine nucleotides. J. biol. Chem., 251,
Comments
Post a Comment
اكتب تعليق حول الموضوع