(Received for publication, November 28, 1994; and in revised form, January 10, 1995)
From the
Most G protein-coupled receptors, including the mammalian
-adrenergic receptor, are endocytosed to an
intracellular, vesicular compartment upon continued exposure to
agonist. The long form of the avian
-adrenergic
receptor, which contains a carboxyl-terminal 59-amino acid extension,
does not undergo agonist-promoted endocytosis. We constructed and
expressed turkey
-adrenergic receptor cDNAs with
regularly spaced carboxyl-terminal truncations and studied their
agonist-promoted endocytosis. Removal of 34-86 amino acids from
the carboxyl terminus of the turkey receptor allowed its efficient
endocytosis, with optimal endocytosis observed upon removal of 59
residues. Removal of only 18 residues allowed some endocytosis. A
receptor that lacks the entire carboxyl-terminal region (124 residues)
was not endocytosed. We also constructed a chimeric hamster
-adrenergic receptor with the added 59-residue
carboxyl-terminal domain of the turkey receptor. The chimera was not
significantly endocytosed. These data indicate that residues
450-465 in the carboxyl-terminal region of the
-adrenergic receptor can act independently to block
agonist-promoted endocytosis and that other carboxyl-terminal
structures nearer to the seventh membrane span are required for
endocytosis.
Upon activation by agonists, G protein-coupled receptors regulate the activity of membrane-bound enzymes, ion channels, and transport proteins by catalyzing the activation of heterotrimeric G proteins ( (1) for review). Prolonged agonist binding also leads to desensitization, a decrease in the magnitude of the response despite the continued presence of agonist. At least three mechanistically and temporally distinct processes contribute to the overall phenomenon of desensitization (see (2) and (3) for review). First, agonist binding induces a rapid uncoupling of receptor from its G protein target. This process involves receptor phosphorylation by one or more protein kinases followed by binding of an inhibitory protein of the arrestin family. Second, agonist binding causes translocation of receptors from the cell surface to an intracellular vesicular compartment, probably endosomes(4, 5) . Such agonist-promoted endocytosis, also referred to in the literature as internalization or sequestration, requires somewhat longer exposure of the receptor to agonist than does the initial uncoupling reaction. Endocytosis has been proposed to play a primary role in the resensitization of receptors(6) . Finally, long term exposure of a receptor to agonist leads to down-regulation, the net loss of receptors from the cell. The mechanism of down-regulation is unclear but probably involves both degradation of endocytosed receptors and decreased receptor synthesis caused in part by destabilization of receptor mRNA(5, 7, 8, 9) .
The
-adrenergic receptors are the most thoroughly studied members of
the G protein-coupled receptor family and have been prototypes in
studies of desensitization. There are three distinct subtypes of the
-adrenergic receptor (designated
,
, and
(10) ). (
)Although these isoforms are quite similar in primary
structures, specificities for ligands and selectivities among G
proteins, they display markedly different desensitization phenotypes.
Upon exposure to agonist, the
-adrenergic receptor is
uncoupled, internalized, and down-regulated(2, 3) . In
contrast, the
-adrenergic receptor does not undergo
any of these modes of desensitization when exposed to
agonists(11) . The desensitization behavior of the
-adrenergic receptor remains unclear. Although there
is general agreement that
-adrenergic receptors
undergo uncoupling in response to agonist, their endocytosis and
down-regulation remain controversial. In tissues, some investigators
have observed down-regulation of
-adrenergic receptors
and others have not, but results have generally not been confirmed for
identical tissues and species in multiple laboratories (see (12) for review). Studies of cultured mammalian cells that
express endogenous
-adrenergic receptors indicate that
these receptors undergo all three desensitization
reactions(13, 14) . However, recent studies of
recombinant human
-adrenergic receptors expressed in
CHW cells found desensitization but no endocytosis or degradation of
receptors(15) . The turkey erythrocyte
-adrenergic receptor also does not display either
endocytosis or down-regulation in erythrocytes, reticulocytes, or
stably transfected L cells(16, 17, 18) .
Several lines of evidence indicate that the cytoplasmic
carboxyl-terminal domain of the -adrenergic receptors at least
partially determines their different desensitization
phenotypes(6, 11, 18, 19, 20, 21, 22, 23, 24) .
Hertel et al.(18) recently found that a spontaneously
mutated avian
-adrenergic receptor, in which 71
carboxyl-terminal amino acid residues were lost, acquired the ability
to undergo both agonist-promoted endocytosis and down-regulation. In
this study, we have examined the role of the carboxyl-terminal domain
of the avian
-adrenergic receptor in modulating its endocytosis
and defined separate sequences that permit and block endocytosis and
shown that the endocytosis-blocking region can function when attached
to other receptors.
The construction of the other
truncated -adrenergic receptor cDNAs used in this
study was described previously(28) . All receptor cDNAs were
inserted into the expression vector pCMV5, and the integrity of the
constructs was confirmed by DNA sequencing and restriction mapping. All
manipulations of recombinant DNA followed standard
procedures(29) .
A series of mutant turkey -adrenergic
receptor cDNAs was constructed in which stop codons were inserted at
evenly spaced intervals throughout the region that encodes the
carboxyl-terminal domain (28) (Fig. 1). These receptors
are referred to as T359, T397, T424, T449, and T465 according to the
numbering of their carboxyl-terminal residues. Each truncated receptor
was expressed stably in L cells, which do not have endogenous
-adrenergic receptors. We also expressed a chimeric receptor
(HTCR) in which the carboxyl-terminal 59 amino acid residues of the
turkey
-adrenergic receptor were appended to the end
of the hamster
-adrenergic receptor. Several clonal
cell lines expressing each receptor were isolated and used in these
experiments. The level of expression of the receptors in the L cell
clones ranged from 90 to 850 fmol/mg of membrane protein. In general,
truncated receptors and the hamster
-adrenergic
receptor were expressed at higher levels than were the wild-type turkey
-adrenergic receptor or the HTCR chimera. Each mutant
receptor displayed the expected size according to photoaffinity
labeling with [
]ICYP-diazirine followed by
dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography
(not shown; see (28) ). When expressed in L cells, each of the
receptors used here mediated activation of adenylyl cyclase by
isoproterenol, as described previously for these and other constructs
(see Table 2, below, and (18) ).
Figure 1:
Structures of the -adrenergic
receptors used in this study. The orientation of structural domains of
the long form (encoded by spliced mRNA(34) ) of the turkey
-adrenergic receptor is shown with reference to the
plasma membrane. The original and novel carboxyl termini are shown as black circles; other residues are shown as open
circles. The mammalian
-adrenergic receptor and
the short form of the turkey
-adrenergic receptor both
terminate at a position homologous to residue
424.
The ability of the
various receptors to undergo agonist-induced internalization was
initially examined by determining the potency with which the
hydrophilic ligand CGP12177 competes with
[I]ICYP for binding to receptors in lysates
prepared either from untreated cells or from cells incubated with
isoproterenol for 20 min. Sequestration in endosomes, which are
relatively inaccessible to hydrophilic ligands, decreases the potency
of inhibition of binding by
CGP12177(31, 32, 33) . As shown previously
and in Fig. 2, treatment with isoproterenol increases the
IC
for CGP12177 binding to
-adrenergic
receptors by about 15-fold. Exposure to isoproterenol does not alter
the IC
for the turkey
-adrenergic
receptor, which is not endocytosed or
down-regulated(16, 28) .
Figure 2:
Effect
of isoproterenol treatment on the sensitivity of -adrenergic
receptors to CGP12177. Untreated cells (
) or cells that were
exposed to isoproterenol for 20 min (
) were lysed, and
[
I]ICYP binding was determined at increasing
concentrations of CGP12177 as described under ``Experimental
Procedures.'' Results are expressed as the percentages of maximal
binding. Each panel shows a single experiment carried out in
triplicate and is representative of at least 3 similar experiments (see Table 1).
In contrast to the
wild-type avian -adrenergic receptor, most of the
truncation mutants were significantly endocytosed, as monitored by
increases in the IC
for CGP12177 ( Fig. 2and Table 1). For T424, whose carboxyl-terminal region is equivalent
in length to that of the
-adrenergic receptor,
isoproterenol was as effective in promoting endocytosis as in the
isoform. Major shifts in the IC
for
CGP12177 were also observed for T398 and T449. Even the removal of only
18 amino acid residues (T465) permitted a small but reproducible
decrease in CGP12177 potency in response to agonist, between 1.2- and
1.8-fold in 6 separate experiments. However, some carboxyl-terminal
structure is apparently required for endocytosis because complete
removal of the carboxyl-terminal domain beyond the palmitoylated
cysteine residue (T359) produced a receptor that was not endocytosed.
Although the truncation mutants and the hamster
-adrenergic receptor were usually expressed at higher
levels than the wild-type turkey
-adrenergic receptor,
endocytosis behavior did not vary notably among L cell clones that
expressed the same receptor at different levels. Endocytosis therefore
does not reflect only the amount of receptor present or the relative
stimulation of adenylate cyclase upon exposure to agonist.
Addition
of the extreme carboxyl-terminal domain of the turkey
-adrenergic receptor to the body of the
receptor markedly diminished the effect of exposure to agonist.
Exposure decreased the IC
for CGP12177 by about 3-fold,
much less than observed with the wild-type
-adrenergic
receptor but still clearly altered. Unlike the other wild-type and
mutant receptors, competition by CGP12177 for ICYP binding to the
chimera was usually multiphasic after exposure of cells to agonist (see Fig. 2, for example), and at least one L cell clone displayed
anomalously high affinity for CGP12177 with or without prior exposure
to isoproterenol (not shown). Thus, addition of the carboxyl-terminal
extension of the turkey
-adrenergic receptor to the
receptor significantly blocked its endocytosis, but
other anomalies in its behavior made the effect less clear cut than was
the case with the truncations.
An alternative assessment of
agonist-promoted endocytosis comes from analysis of the distribution of
receptors among cellular membrane fractions after their separation by
sucrose density gradient centrifugation. As shown in Fig. 3, the
endocytosis of -adrenergic receptors is reflected by
movement of receptors from a high-density peak, characteristic of
plasma membrane fragments, to a low-density peak that is characteristic
of endosomes (4, 38) . In addition to the change in
sedimentation, receptors in the endosome fraction are relatively
insensitive to the hydrophilic
-adrenergic antagonist CGP12177,
presumably because their ligand-binding sites face the lumen of the
endosomes. Thus,
-adrenergic receptors from cells that
were not exposed to agonist fractionated primarily at high density, and
ICYP binding in this fraction was blocked 90% by 1 µM CGP12177. In contrast, 55% of the
-adrenergic
receptors from cells that were exposed to isoproterenol were found in
the endosome fraction. For these receptors in the low-density peak,
CGP12177 blocked ICYP binding by only 18%. As shown previously (18,
confirmed in Fig. 3), the turkey
-adrenergic
receptor did not undergo agonist-promoted endocytosis according to this
assay. The turkey
-adrenergic receptors remained in
the high-density peak after treatment of cells with isoproterenol, and
those receptors remained sensitive to CGP12177. According to this
assay, T424 receptors behaved like mammalian
-adrenergic receptors: they moved to the endosome
fraction and were protected from CGP12177. In contrast, the chimeric
receptors behaved like turkey
-adrenergic receptors
and remained in the plasma membrane fraction.
Figure 3:
Effect of isoproterenol treatment on
sedimentation of wild-type and mutant -adrenergic receptors.
Untreated cells (squares) or cells exposed to isoproterenol
for 20 min (circles) were lysed and lysates were fractionated
on sucrose gradients as described under ``Experimental
Procedures.'' The binding of [
I]ICYP to
receptors in gradient fractions was determined in the presence (open symbols) or absence (closed symbols) of
30-60 nM CGP12177. Data points are averages of
triplicate assays performed in the presence and absence of 1 µM (-)-propranolol to determine specific receptor
binding.
As summarized in Fig. 4for all mutant and wild-type receptors, the distal
carboxyl-terminal domain of the turkey -adrenergic
receptor predominantly determined the migration of receptors from the
plasma membrane fraction to the endosome fraction when cells were
exposed to isoproterenol. Fig. 4shows the ratios of receptors
found in endosomes and plasma membranes with and without exposure of
the cells to isoproterenol. For every construct tested, fewer than 15%
of the receptors were endosomal unless the cells were exposed to
isoproterenol. After exposure to isoproterenol, both the hamster
-adrenergic receptor and three truncated turkey
-adrenergic receptors (T397, T424, and T449) were
largely endocytosed. The wild-type
-adrenergic
receptor and the chimera were not detectably endocytosed. Only a
minority of the T359 and T465 receptors were found in endosomes, but a
low level of endocytosis was noted reproducibly. Thus, the intact
carboxyl terminus of the turkey
-adrenergic receptor
can block endocytosis even of a
-adrenergic receptor,
and the behavior of the truncation mutants indicates that 18 amino acid
residues is the minimum amount necessary for this function. Note that
the calculations used to generate the data shown in Fig. 4underestimate the sizes of both pools of receptors.
However, calculating the ratios without reference to sensitivity to
CGP12177 did not alter the overall pattern depicted in the figure.
Figure 4:
Effect of isoproterenol treatment on the
distribution of wild-type and mutant -adrenergic receptors between
endosomal vesicles and plasma membrane. Control cells (hatched
bars) or cells exposed to isoproterenol (INE) for 20 min (open bars) were lysed and analyzed by sucrose density
gradient centrifugation as described in the legend to Fig. 3and
under ``Experimental Procedures.'' Receptors in the endosomal
vesicles were defined as [
I]ICYP binding
activity in the low-density fraction that was not blocked by
30-60 nM CGP12177. Receptors in the plasma membranes
were defined as [
I]ICYP binding activity in the
high density fraction that was blocked by CGP12177. Data shown are
means of 2, 3, 5, 4, 3, 3, 4, or 4 complete experiments (left to right). Error bars indicate S.E. values for
isoproterenol-treated cells only. S.E. values for control cells varied
from 0.01 to 0.07 (not shown).
Although the turkey -adrenergic receptor is not
endocytosed or down-regulated in response to agonist, it does undergo
homologous desensitization in the form of uncoupling from G protein (16) . The data in Table 2indicate that truncating even
the entire carboxyl-terminal region, in the T359 mutant, did not
markedly diminish uncoupling. Although desensitization varied among
experiments, its extent was generally 25-50% for both wild-type
and mutant receptors. Likewise, attachment of the carboxyl-terminal
region of the turkey receptor to the hamster
-adrenergic receptor did not markedly influence its
desensitization.
Agonist-promoted internalization is a common property in the
G protein-coupled receptor family, but the precise sequence motifs
within these receptors that control internalization are not well
defined. Diverse sequences in the cytoplasmic end of the seventh helix,
the carboxyl-terminal domain, and the third cytoplasmic loop have all
been implicated in initiating, inhibiting, or allowing endocytosis of
receptors(2, 3) . The present study defines two
regions in the carboxyl-terminal domain of the avian
-adrenergic receptors that are involved with its
endocytosis. First, the endocytosis-incompetent phenotype of the T359
receptor confirms previous indications that the region just beyond the
palmitoylated cysteine residue is required for endocytosis in response
to agonist(6, 22, 24) . Second, the region
between residues 450 and 465 blocks endocytosis in a receptor that is
otherwise endocytosis-competent. The existence of a discrete
endocytosis-blocking domain has been defined only in the avian
-adrenergic receptor(18, 28) , and
this domain accounts for the inability of this receptor to be
endocytosed(16, 17, 18) .
The mechanism
whereby the carboxyl-terminal region prevents endocytosis remains
unclear. When these same truncation mutants are expressed in Sf9 cells,
we found that several other phenotypes coincided with restoration of
endocytosis(28) . Although their intrinsic affinities for
-adrenergic ligands are unaltered, the truncation mutants are more
active stimulators of adenylyl cyclase in Sf9 cell membranes than are
wild-type receptors. They stimulate somewhat in the absence of agonist,
produce greater maximal activities in the presence of agonists, and are
strikingly more sensitive to weak partial agonists. Their affinities
for agonists are also more sensitive to guanine nucleotides. Last, the
truncated receptors are expressed at higher levels and are more easily
solubilized by mild detergents than are the wild-type receptors, which
predominantly remain in the pellet after extraction with digitonin,
dodecyl maltoside, or several other detergents(28) . These
diverse effects led us to suggest that the carboxyl-terminal region may
anchor the receptors either to cytoskeleton or to some other
detergent-insensitive structure, such as caveolae. Such anchorage would
both prevent their movement to coated pits, the site of endocytosis,
and limit their access to G proteins. Several G proteins are found in
caveolae, and the behavior of several G protein-coupled receptors is
strongly influenced by drugs that interact with the
cytoskeleton(35, 36, 37) .
The putative
anchoring region at the carboxyl terminus of the turkey
-adrenergic receptor evidently constitutes an
independent structural and functional domain within the protein. Its
removal from the turkey receptor does not damage receptor function, and
its addition to the hamster
-adrenergic receptor
confers the phenotypes characteristic of the turkey receptor. According
to analysis by sucrose density gradient centrifugation, the HTCR was
not endocytosed at all in response to agonist ( Fig. 3and Fig. 4). Its behavior in the CGP12177 competition experiments
also indicated marked blockade of endocytosis, although interpretation
of the data was not straightforward (Fig. 2, Table 1).
From the biphasic shape of the competition curve shown in Fig. 2and observed in other experiments, it appears that
endocytosis of most of the receptors was totally blocked, but that a
minority of HTCRs were protected from CGP12177. This might indicate
that a few HTCRs are mis-sorted in the cell or that some undergo
intracellular proteolysis to lose the anchorage domain.
The
functional independence of the carboxyl-terminal anchorage domain is
particularly striking because it is encoded by a 3` exon separate from
the exon that encodes the rest of the receptor. The receptor RNA is
alternatively spliced to yield either the long (full-length) receptor
or a short receptor that is equivalent to T424(34) . Thus, a
cell can use a single gene to express -adrenergic
receptors that either are or are not endocytosed in response to
continued exposure to agonist.
The experiments reported here specify
a small region of the turkey -adrenergic receptor that
inhibits agonist-promoted endocytosis. Removal of only 18
carboxyl-terminal amino acids permitted some endocytosis, as measured
either by increased protection of the receptors from inhibition by the
hydrophilic antagonist CGP12177 (Fig. 2, Table 1) or by
movement of receptors to small, sealed vesicles (Fig. 4).
Complete restoration of endocytosis was observed in the T449 mutant, in
which 34 amino acid residues were removed, and little further effect
was observed in T424 or T397. The region between 450 and 470 is
therefore primarily responsible for anchoring the receptor in the
plasma membrane. The small amount of endocytosis of the T465 mutant
might reflect partial loss of the relevant structure, but it seems more
likely that the important region lies between residues 449 and 465 and
that the T465 phenotype reflects improper folding of that region. This
conjecture is intuitively supported by the finding that residues
449-467 are conserved in the 3` open reading frames of the monkey
and rat
-adrenergic receptor genes(34) . The
sequence of this region is unremarkable and has only slight homology to
other known proteins(34) . Regardless, the mapping of the
anchorage domain in the present study is now fine enough to allow
site-directed mutagenesis to determine its important structures.
In
addition to their failure to endocytose, turkey
-adrenergic receptors are not down-regulated in
response to agonist(16, 17, 18) . However, a
spontaneous mutant that has lost the carboxyl-terminal domain is both
efficiently endocytosed and down-regulated(18) . This finding
suggests that the anchorage domain blocks down-regulation because it
blocks endocytosis, a likely precursor to receptor
degradation(2) . In many cells, however, down-regulation
reflects both synthesis and degradation of receptors. Because the
synthesis of recombinant receptors under the control of plasmid-born
regulatory elements may not provide a valid image of physiological
regulation, we have not studied down-regulation of the truncation
mutants. On the other hand, the retention of agonist-promoted
desensitization by all of the mutants (Table 2), which is
characteristic of avian
-adrenergic receptors, argues
that the carboxyl terminus of the
-adrenergic receptor
is not required for receptor-G protein uncoupling. The
carboxyl-terminal domain thus restricts long term removal of receptors
by endocytosis and degradation while allowing desensitization mediated
by uncoupling from G protein.