(Received for publication, May 8, 1995; and in revised form, July 17, 1995)
From the
Following agonist stimulation, many receptors are rapidly
internalized from the plasma membrane via a mechanism which presumably
involves recognition motifs within the cytoplasmic domains of the
receptor. We have previously demonstrated (Thomas, W. G., Thekkumkara,
T. J., Motel, T. J., and Baker, K. M. (1995) J. Biol. Chem. 270, 207-213) that truncation of the angiotensin II
(AT) receptor, to remove 45 amino acids from the
cytoplasmic tail, markedly reduced agonist stimulated receptor
endocytosis. In the present study, we have stably and transiently
expressed wild type and carboxyl terminus mutated AT
receptors in Chinese hamster ovary cells to identify regions and
specific amino acids important for this process. Wild type AT
receptors rapidly internalized (t = 2.5 min; Y
= 76.4%) after AII stimulation. Using
AT
receptor mutants, truncated and deleted at the carboxyl
terminus, two distinct regions important for internalization were
identified: one membrane proximal site between residues 315-329
and another distal to Lys
, within the terminal 26 amino
acids. Point mutations (Y302A, Y312A, L316F, Y319A, and K325A) were
performed to identify residues contributing to the membrane proximal
site. Mutation of Y302A, Y312A, and K325A had little effect on the rate (t = 4.3, 2.8, and 2.8 min) and maximal amount (Y
= 81.7, 67.8, and 73.5%) of AII
induced internalization. In contrast, L316F and Y319A mutations
displayed an approximately 2.5-fold reduction in rate (t = 6.1 and 6.2 min) and L316F a decreased maximal level (Y
= 38.1 and 71.4%, respectively)
compared to wild type. Interestingly, Leu
and
Tyr
are closely aligned within the hydrophobic aspect of
a putative amphipathic helix, possibly representing an internalization
motif for the AT
receptor. We conclude that the
AT
receptor does not use the NPXXY
(NPLFY
) motif, first described for the
-adrenergic receptor, to mediate agonist stimulated
endocytosis. Rather, two distinct regions of the carboxyl terminus are
utilized: one involving hydrophobic and aromatic residues on a putative
-helix and another serine/threonine-rich domain.
Endocytosis of cell surface receptors is a ubiquitous eukaryotic process. It permits the internalization of extracellular nutrients (e.g. cholesterol via low density lipoprotein receptors), serves to dampen cell responses by removing ligand-activated receptors from the cell surface, and mediates cellular resensitization by recycling functional receptors to the cell surface(1, 2, 3, 4, 5, 6) . Although some endocytosis occurs constitutively, the rate is increased dramatically following binding of many extracellular ligands to their cognitive transmembrane receptors. This process is homologous in that usually only receptors for the stimulating ligand are internalized, and of those, only receptors occupied by ligand are targeted. Homologous endocytosis predicts that receptors inherently contain within their structures and sequences determinants for internalization, and that these determinants are concealed or remain latent until agonist binding. Thus, it is generally assumed that within the cytoplasmic domains of receptors are one or more amino acid codes, or motifs, that are recognized by adaptor complexes which mediate the selective recruitment of receptor-ligand complexes into primarily clathrin-coated vesicles (3, 5, 7, 8) or non-coated vesicles and caveolae (discussed in (9) ). Commonly, these motifs include aromatic (predominantly tyrosine) and hydrophobic amino acids, and while phosphorylation of these crucial tyrosines does not necessarily drive the internalization process, phosphorylation at other sites may initiate allosteric changes within the receptor responsible for unmasking endocytotic codes (10, 11, 12) .
Angiotensin II (AII) ()is a peptide hormone with important actions on blood
pressure regulation, water and salt balance, neuromodulation, and
cellular growth(13) . Two major types of AII receptors, termed
AT
and AT
, and two subtypes of the AT
receptor, termed AT
and AT
, have been
identified(14) . These receptors are all members of the seven
transmembrane, guanyl nucleotide-binding protein (G-protein)-coupled
receptor (GPCR) superfamily, but the AT
receptor, which
contains 359 amino acids including approximately 54 amino acids as a
carboxyl-terminal cytoplasmic tail, is the principal mediator of the
biological actions of AII. AII receptors present on cells cultured from
various tissues (15, 16, 17, 18, 19) and
cloned AT
and
AT
(20, 21, 22, 23, 24) ,
but not AT
(23) , receptors expressed in cell
systems, rapidly internalize upon AII binding. Internalization of
AII
receptor complexes is independent of G-protein coupling (20, 22) and occurs via a clathrin-dependent
process(20) , which is the primary route for many, but not all,
GPCRs. While the exact cellular processes and receptor motifs that
control AT
receptor endocytosis are unknown, the AT
carboxyl terminus contains four candidate tyrosine residues
(Tyr
, Tyr
, Tyr
, and
Tyr
). Interestingly, Tyr
in the motif
NPXXY is analogous to Tyr
shown to be important
for
-adrenergic receptor internalization(25) ,
but the contribution of Tyr
to AT
receptor
endocytosis has not been reported. The serine/threonine-rich portion of
the carboxyl terminus has been implicated in AT
receptor
internalization(23) , and we recently demonstrated that
truncation of the AT
receptor to delete the
carboxyl-terminal 45 amino acids markedly reduced agonist-mediated
endocytosis(24) . In the present study, we have stably and
transiently expressed in CHO-K1 cells AT
receptors
containing truncation, deletion, and point mutations of the cytoplasmic
tail and determined AII stimulated endocytosis. Our data implicate two
separate domains within the carboxyl-terminal region of the AT
receptor in endocytosis and identify key amino acids involved in
this process.
To
truncate the AT receptor after Lys
, a
1126-base pair fragment was PCR amplified from the clone pB2/AT
using a sense primer (5`-GTAAAGCTTAAGTGGATTTCG-3`) and antisense
primer (5`-GGTAGAAAGCTTGCTCTATTTCGTAGAC-3`), incorporating HindIII restriction sites (underlined) and an antisense stop
codon (italics) after Lys
. The amplified DNA was
restriction digested with HindIII and subcloned directly into
the pRc/CMV vector (pRc/TK333). Sequencing with the Sequenase 2.0 kit,
as described previously(26) , confirmed the correct orientation
and the entire coding region. Deletion of 15 amino acids (Glu
through Ser
) from the full cytoplasmic tail was
constructed in two steps. First, a 1012-base pair fragment was PCR
amplified from the clone pB2/AT
using the same sense
primer as above and an antisense primer
(5`-TTTCAGGAGCTCGAGGAAATACTT-3`), incorporating a XhoI site
(underlined). The PCR fragment, coding for amino acids 1-314, was
restriction digested with HindIII and XhoI and
subcloned into pBluescript II (pB/1-314). Second, a DNA fragment,
coding for amino acids 330-359, was PCR amplified from
pB2/AT
using a sense primer
(5`-TCCTGTCTACGAAAATGAGCACG-3`) and an antisense primer
(5`-CTACAGTCTGATGGGCCCATTTTTCTGCTTAG-3`), incorporating an ApaI restriction site (underlined). pB1-314 was digested
with XhoI, blunted using mung bean nuclease and a second
restriction digest performed with ApaI. The second PCR product
was digested with ApaI and ligated into the modified
pB1-314. The integrity of the deletion mutant construct was
confirmed by sequencing and then released by an ApaI/HindIII digest for cloning into pRc/CMV
(pRc/Del315-329).
Point mutations were introduced into the
AT receptor using the PCR-based method of Ito et
al.(27) . Tyrosines at positions 302, 312, and 319 and
lysine at position 325 were individually mutated to alanine, and
leucine at position 316 was mutated to phenylalanine, using the
following primers (mutated bases are underlined): Y302A,
5`-AACCCTCTGTTCGCCGGCTTTCTG-3`; Y312A, 5`-GAAATTTAAAAAGGCTTTCCTCCAG-3`;
L316F, 5`-GTATTTCCTCCAGTTCCTGAAATAT-3`; Y319A,
5`-CAGCTCCTGAAAGCTATTCCCCCAAAG-3`; K325A,
5`-CCCCCAAAGGCCGCGTCCCACTCA-3`.
All mutated PCR fragments were cloned into pBluescript II and the entire coding region and the relevant mutation confirmed by sequencing. Mutated receptor sequences were restriction digested with HindIII and subcloned into pRc/CMV at the same site to yield the expression plasmids pRc/Y302A, pRc/Y312A, pRc/L316F, pRc/Y319A, and pRc/K325A.
The authenticity of the
data obtained from the clonal lines was also confirmed using transient
transfections. For this, CHO-K1 cells at 80% confluence, in 35-mm
dishes, were transfected using the lipofectamine method, as described
by the manufacturer (Life Sciences). Cells were washed in OPTI-MEM, and
for each well 1 µg of plasmid DNA was mixed with 8 µl of the
lipofectamine reagent (2 mg/ml) in 200 µl of OPTI-MEM for 20 min.
The mixture was diluted to 1 ml with OPTI-MEM and placed on the washed
cells, which were returned to the incubator for 5 h. The
DNA/lipofectamine solution was aspirated and replaced with 3 ml of
complete media. Cells were cultured for 48 h and internalization assays
performed. For each transfection, I-AII binding assays
were performed in parallel on some wells to confirm transfection
efficiency and receptor expression. The level of receptor expression,
extrapolated by assuming similar dissociation constants established for
the clonal cell lines, was approximately 400-600 fmol/mg protein
for the wild type receptors and 100-400 fmol/mg protein for the
mutants.
cDNAs encoding wild type and mutated rat AT receptors were subcloned into the pRc/CMV mammalian expression
vector and transfected into CHO-K1 cells. The various constructs are
depicted in Fig. 1. Colonies resistant to neomycin (G418) were
selected and propagated, and individual clonal lines expressing
functional receptors were selected by the ability to bind
I-AII. To determine if the expressed mutated receptors
displayed affinities comparable to the wild type receptor, competition
binding studies were performed. As summarized in Table 1, the
wild type and assorted truncation, deletion, and point substitution
mutants all bound
I-AII with high affinity (K
approximating 1 nM) indicating that
these receptors attain a conformation necessary for high affinity
recognition of AII. Dissociation constant values in the nanomolar range
compare well with those previously reported for AT
receptors expressed in cells and tissues(33) . The level
of receptor expression for the mutated receptors ranged between
145-840 fmol/mg protein (Table 1).
Figure 1:
Schematic
representation of the wild type rat AT receptor and
mutations to the carboxyl-terminal region. The top of the figure
illustrates the predicted seven transmembrane topology of the receptor,
with residues 298 through 359, which comprise the end of the seventh
transmembrane segment and the entire carboxyl-tail, shown as open
circles. The bottom of the figure is the one-letter amino
acid representation for residues 298 to 359 for the wild type,
truncated, deleted, and point-mutated constructs. The end of the
seventh transmembrane segment is indicated (TM7) on the wild
type sequence. For point mutations, the specific residue changed is underlined and the substitution indicated
above.
Using the appearance
of acid-resistant radioactivity, after addition of I-AII
at 37 °C as an index of receptor internalization, we observed that
wild type AT
receptors expressed in CHO-K1 cells underwent
rapid endocytosis from the plasma membrane (Fig. 2). Shown in Fig. 2is the internalization profile for four separate clonal
lines (T3, T5, T11, and T24) expressing the wild type receptor.
Although the level of expression in these clones varies over a 10-fold
range (T3, 3400 fmol/mg protein(26) ; T24, 667 fmol/mg
protein(24) ; T11, 330 fmol/mg protein; T5, 310 fmol/mg
protein), the kinetics of
I-AII internalization, fitted
as one phase exponential associations, were similar for all four clonal
lines (T3, t 2.1 min, Y
80.9%; T5, t 2.3 min, Y
79.4%; T11, t 2.3
min, Y
80.6%; T24, t 2.5 min, Y
76.4%). This observation indicates that the
level of receptor expression does not influence the degree of
agonist-stimulated endocytosis and therefore internalization parameters
for the mutant receptors can be directly compared despite varying
levels of receptor expression (145-840 fmol/mg protein). One of
the wild type receptor expressing clones, T24, was chosen for
comparison with cells expressing mutated receptors. Also shown in Fig. 2is the markedly reduced internalization (Y
16.2%) of TL314, confirming our previous
observation (24) that the carboxyl-terminal 45 amino acids of
the AT
receptor play a critical role in endocytosis. It is
noteworthy that removal of 45 amino acids from the cytoplasmic tail did
not prevent high affinity binding of AII, coupling to G-proteins, and
appropriate activation of conventional signaling pathways(24) .
Figure 2:
Endocytosis of the AT
receptor is independent of receptor density but dependent upon an
intact carboxyl terminus. CHO-K1 cells stably expressing varying
densities of wild type AT
receptor (T3, 3400
fmol/mg protein, filled circles; T5, 310 fmol/mg
protein, open circles; T11, 330 fmol/mg protein, filled squares; T24, 667 fmol/mg protein, open
squares) or a carboxyl-truncated AT
receptor (TL314, crosses) were incubated with
I-AII for 2-20 min at 37 °C. At the indicated
times, surface-bound and internalized
I-AII were
determined by acid washing as described under ``Experimental
Procedures.'' An index of internalization was calculated by
expressing the internalized radioactivity (acid-resistant) as a
percentage of the total binding (acid-resistant plus acid-susceptible).
Data are means ± S.D. for three (TL314), six (T3, T5, and T11)
or nine (T24) determinations.
To further define sites important for internalization (within this
carboxyl-terminal region), we evaluated the capacity of another
truncated receptor, TK333, to undergo AII-mediated endocytosis (Fig. 3). This mutant displayed a slower (t 6.1 min)
and reduced amount (Y 46.3%) of internalization
compared to the wild type T24 (t 2.5 min, Y
76.4%), implicating the terminal 26 amino acids in the
internalization process. However, the level of internalization observed
for TK333 was consistently greater than that observed for TL314 (Y
46.3 versus 16.2% (see Fig. 2)), which suggested that the region between Leu
and Lys
also contained an endocytotic determinant.
To confirm this, a deletion mutant was constructed in which the
terminal 30 amino acids(330-359) were fused to the first 314
residues, deleting amino acids 315-329. As shown in Fig. 3,
I-AII was slowly (t = 8.6
min) internalized by this mutant, to a degree (Y
= 40.9%) slightly less than that displayed by TK333. These
observations suggest that there are two sites important for endocytosis
within the carboxyl terminus of the AT
receptor; one
distal to Lys
and another in the region 315-329.
Figure 3:
Two separate regions of the AT receptor carboxyl terminus are important for endocytosis.
Internalization kinetics for the wild type AT
receptor (filled circles) and truncated (TK333, open
circles) and deletion (Del315-329, squares) mutants were obtained as described in the legend to Fig. 1. Data are the means ± S.D. from three separate
experiments performed in triplicate.
While these observations were being made, Hunyady et al.(23) reported the inhibition of AT receptor
internalization by mutation of amino acids within a carboxyl-terminal
serine/threonine-rich region, in particular, residues
Ser
, Thr
, and Leu
. The
location of these residues distal to our truncation of the receptor at
Lys
explained the diminished internalization observed for
TK333. We reasoned, however, that this ``STL motif'' alone
was not sufficient to direct internalization because our deletion
mutant (Del315-329) displayed markedly reduced endocytosis (t = 8.6 min, Y
= 40.9%),
despite the presence of this tripeptide sequence as well as other
contributing residues, Thr
and Ser
(23) . Thus, we focused on identifying specific residues
in the more proximal regions of the carboxyl-tail. The first point
mutants constructed were Tyr
to alanine (Y302A) and
Lys
to alanine (K325A). The rationale for altering these
specific residues was that Tyr
is a highly conserved
residue in many G-protein-coupled receptors and because the analogous
Tyr
in the
-adrenergic receptor has been
identified as a key residue in the internalization
process(25) . The K325A mutant was engineered because: 1) this
amino acid is present in the configuration AKS which is similar to the
internalization motif DAKSS(34) , with its crucial central
lysine, found in the yeast
-factor receptor, also a seven
transmembrane spanning protein like AT
; and 2) Lys
is located within the region(315-329) identified as
important for AT
receptor endocytosis. Shown in Fig. 4are the internalization profiles for Y302A and K325A
compared to the wild type receptor. The internalization kinetics for
the K325A mutant were equivalent (t 2.8 min, Y
73.5%) to the wild type receptor, suggesting
that this residue is not critical for internalization. The Y302A
mutation had little effect on AT
internalization; the rate
was slightly reduced (t 4.2 min), but the maximal level of
receptor endocytosis was comparable (Y
81.7%) to
wild type.
Figure 4:
Single point mutants Y302A and K325A and
the wild type AT receptor display similar endocytosis.
Internalization kinetics for CHO-K1 cells stably expressing the wild
type (filled circles), Y302A (open circles), and
K325A (open squares) AT
receptors were determined
as described in the legend to Fig. 1. Data are the means
± S.D. from three separate experiments performed in
triplicate.
Given the prevalence of tyrosine residues in the
endocytotic motifs for many receptors(3, 5) , we next
mutated Tyr and Tyr
separately to alanines.
Also, for some receptors (e.g. insulin(35) ),
dileucine residues within the cytoplasmic regions are important for
efficient receptor internalization. Since the AT
receptor
carboxyl terminus includes a dileucine pair
(Leu
Leu
) and because these leucines are
located between residues 315-329, we decided to test the
involvement of this putative motif by mutating Leu
to
another hydrophobic residue, phenylalanine (L316F). Shown in Fig. 5are the internalization profiles for Y312A, L316F, and
Y319A. Mutation Y312A internalized at a rate (t 2.8 min) and
to a degree (Y
67.8%) comparable with the wild
type receptor. Mutation L316F displayed a significantly reduced
capacity for endocytosis with a 2.5-fold reduction in rate (t 6.1 min) and a diminished maximal level (Y
38.1%). The internalization rate (t 6.2 min) for Y319A
was also reduced, but the Y
at 71.4% was similar
to that of the wild type receptor.
Figure 5:
Comparison of endocytosis for wild type
AT receptors and Y312A, L316F and Y319A mutants.
Internalization kinetics for CHO-K1 cells stably expressing the wild
type (filled circles), Y312A (open circles), L316F (open squares), and Y319A (filled squares) AT
receptors were determined as described in the legend to Fig. 1. Data are the means ± S.D. from three separate
experiments performed in triplicate.
The results presented above
implicate Leu and Tyr
in the control of
AT
receptor endocytosis. These observations were confirmed
in other clonal lines expressing these two mutations, but as further
confirmation that our observations are not the result of aberrations
arising from clonal selection, we performed transient transfections for
selected constructs. CHO-K1 cells were transfected using lipofectamine
reagent with pRc/AT
(wild type) or DNA for mutated
receptors (pRc/TL314, pRc/TK333, pRc/Del315-329, pRc/L316F, and
pRc/Y319A), and 48 h after transfection internalization assays were
performed. The amount of internalization was determined at 10 min, the
time of maximal endocytosis for the wild type receptor. As shown in Fig. 6, the results of transient assays closely mirrored the
data obtained with the clonal cell lines. Thus, compared to wild type
(66.8%), TK333 (28.1%) exhibited reduced endocytosis, but not to the
level displayed by TL314 (9.6%) and Del315-329 (6.8%). Moreover,
the single point mutations L316F (26.8%) and Y319A (46.1%) showed
diminished internalization which was comparable to that observed in
clonal counterparts at 10 min.
Figure 6:
Endocytosis of the wild type AT receptor and various truncated, deleted, and point mutants
following transient transfection in CHO-K1 cells. CHO-K1 cells were
transfected with plasmid DNA encoding wild type
(pRc2A/AT
), truncated (pRc/TL314 and pRc/TK333), deleted
(pRc/Del315-329), and point mutated (pRc/L316F and pRc/Y319A)
AT
receptors, and 48 h later internalization assays were
performed as described under ``Experimental Procedures.'' The
percentage internalization for mutant receptors was determined 10 min
after addition of
I-AII at 37 °C, a time where
internalization of the wild type was maximal. Data are the means
± S.D. for duplicate determinations of the number of experiments
shown on the graph, except for TK333 which is the mean only of data
(28.1 and 28.2%) from two experiments performed in
duplicate.
The rapid (t 2.5 min) internalization of
AII
AT
receptor complexes observed in this study
reflects the dramatic agonist-stimulated endocytosis observed with many
GPCRs, including endogenous and cloned AII
receptors(15, 16, 17, 18, 19, 20, 21, 22, 23, 24) .
It is generally accepted that internalizing receptors contain motifs or
``codes'' (3, 5) that are recognized by
adaptor proteins which mediate the selective aggregation and
association of ligand-bound receptors into clathrin-coated pits. These
pits then invaginate to form vesicles which fuse with
endosomes/lysosomes where low pH dissociates ligand from receptor. The
receptor and ligand are then either degraded, or the receptor is
recycled back to the cell membrane and the ligand, in the case of AII,
may serve to activate putative cytoplasmic (36) or nuclear (37) receptors. While alanine scanning has identified some
residues that are required for efficient internalization of some GPCRs,
no consensus motif has emerged. In this study, we have identified two
disparate regions of the AT
receptor involved in
AII-stimulated endocytosis. One region is approximately 10 amino acids
from the cytoplasmic face of the membrane and involves the hydrophobic
residue Leu
and the aromatic Tyr
, while the
other site is more distally located within the serine/threonine-rich
region of the receptor tail.
Our observation that two distinct
regions of the cytoplasmic tail are important for internalization
corroborates accumulating evidence for multiple endocytotic codes in
other receptors. For example, the cytoplasmic domain of the insulin
receptor contains two tyrosine-based internalization motifs (GPLY and
NPEY) and a juxtamembrane dileucine motif (EKITLL)(35) .
Similarly, the carboxyl-terminal region of the epidermal growth factor
receptor contains at least three endocytotic codes as well as distinct
regions for lysosomal targeting(11) . Endocytosis of the
mannose-6-phosphate receptor requires two separate regions containing
aromatic residues(38) . In addition to receptors with two or
more positive internalization codes, some receptors also display both
positive and negative regulatory
motifs(39, 40, 41) . Our observation of two
positive internalization ``motifs'' for the AT receptor most readily compares to another GPCR, the
thyrotrophin-releasing hormone receptor(42) . In this receptor,
two dissimilar positive sites are also important for endocytosis: one
membrane proximal and another more distal site involving serine and
threonine residues. The similarity of AT
and
thyrotrophin-releasing hormone receptors also includes the spacing from
the membrane of the first site (
10 amino acids) and distance of
the two sites from each other in the cytoplasmic tail (
20 amino
acids). Whether this arrangement is exploited by other GPCRs and the
possible interaction between these two sites and the endocytotic
machinery remains to be established.
In the pursuit of a universal
GPCR internalization motif, Barak et al.(25) recently
reported that Tyr, located near the junction of the
seventh transmembrane segment and the cytoplasmic tail, is a key
residue for the rapid internalization of
-adrenergic
receptors. This observation has particular relevance because many GPCRs
possess an analogous tyrosine residue within a highly conserved
NPX
Y motif (where X is any amino acid).
This motif is similar to the NPXY internalization motif, first
described for the low density lipoprotein receptor(43) , which
is present within the cytoplasmic domains of many receptors. However,
we observed in this study that mutation of the corresponding residue
(Tyr
) in the AT
receptor had little effect
on the kinetics of AII-stimulated internalization. This observation
suggests that Tyr
(NPLFY
) does not input
significantly into AII-stimulated AT
receptor endocytosis,
and given that a similar motif (NPFLY
) is maintained in
the AT
receptor, which does not internalize(23) ,
provides further evidence that other sites are responsible for
internalization of AT
receptors. Indeed, the
NPX
Y motif may only function for a subset of GPCRs
because the comparable tyrosine mutation in the gastrin releasing
peptide receptor was also ineffective in modulating
endocytosis(44) , and because a subgroup of GPCRs (e.g. receptors for secretin, glucagon, parathyroid hormone, calcitonin,
etc.) which lack the NPX
Y motif display rapid
internalization in response to agonist.
Our observation that one of
the two sites important for internalization is located distal to
Lys within the terminal 26 amino acids complements the
recent findings of Hunyady et al.(23) . Using serial
truncations and point mutations of the AT
receptor, they
identified an important contribution of residues in the region
Thr
-Ser
, with a major involvement of the
tripeptide sequence
Ser
Thr
Leu
. However, our
observation that TK333 (removal of 26 amino acids) still displayed some
internalization, at a level greater than observed for TL314 (deletion
of 45 amino acids), suggested that more proximal sites in the carboxyl
terminus were also involved. We therefore engineered a deletion mutant
in which the terminal 30 amino acids of the AT
receptor,
including all residues (Thr
-Ser
) identified
as important by Hunyady et al.(23) , were joined to
the receptor truncated after Leu
. We hypothesized that if
the Thr
-Ser
region was sufficient for
endocytosis, then attachment of these sequences to the noninternalizing
truncated receptor would rescue the internalization response. Such
transplantation of putative internalization motifs onto internalization
incompetent receptors has been previously used to positively identify
such sites(45, 46, 47) . In contrast, we
noted that the endocytosis of this deletion mutant, while apparent, was
markedly reduced in comparison to wild type and was slightly less than
TK333. These results suggest that the region between Gln
and Ser
is as important as the more distal STL
motif and also raise the possibility that the two regions may be
cooperative for endocytosis.
Our observation of a second site within
the AT receptor tail is novel. Moreover, we have
identified a key role for residues Leu
and Tyr
which is consistent with the use of aromatic and hydrophobic
residues for internalization motifs(3, 5) . Single
point mutation of Leu
potently inhibited AII-stimulated
endocytosis with equivalent efficacy to the large deletion
(Del315-329) and truncation (TK333) mutants. Thus, this and
surrounding residues represent attractive candidates for the AII
internalization motif. What is particularly interesting is that
Leu
is part of a dileucine pair
(Leu
Leu
), which is a motif utilized by
insulin receptors (35) and the IgG receptor Fc
RI (48) for endocytosis. A role of Leu
Leu
is supported by the observation that this dileucine pair is not
conserved in the noninternalizing rat AT
receptor(23) . Further studies will be required to
determine whether this region is the actual motif engaged by the
endocytotic machinery or whether the deleterious effect of this
mutation is secondary to conformational changes which prevent
interaction with other sites.
Based on computer modeling and NMR
analysis of peptide sequences, internalization motifs are proposed to
involve a type I -turn with an exposed aromatic (tyrosine)
residue(5) . We have used the computer modeling method of Rost
and Sander (30, 31, 32) to predict the
secondary structure of the cytoplasmic tail of the AT
receptor. The seventh transmembrane segment of the 359 amino acid
AT
receptor exits the cell membrane around Leu
leaving a cytoplasmic carboxyl terminus of 54 amino acids. The
region immediately adjacent to the membrane, between Lys
and Ile
, displayed a high probability of forming an
-helix which was terminated by consecutive proline residues
(Pro
Pro
), while the remainder of the
cytoplasmic tail was predicted to form an extended loop structure. As
shown in Fig. 7, plotting residues Lys
through
Ile
as a helical wheel reveals an amphipathic nature to
this putative helix. The helix divides symmetrically into one side
containing exclusively hydrophobic and aromatic side chains (Leu, Ile,
Phe, and Tyr) with the other half containing mostly basic (Lys) amino
acids. It is interesting that residues Leu
and
Tyr
, which we have shown are important for AT
receptor endocytosis, are closely positioned (within 60°)
toward the end of this helix. The hydrophobic residues on this aspect
would be expected to be protected from the aqueous environment by
burying them away from the surface. Alternatively, these hydrophobic
residues may be concealed through interaction with other proteins,
since amphipathic helices are often involved in protein-protein
interactions. Presumably, ligand binding leads to allosteric changes
which expose these sites to the endocytotic machinery.
Figure 7:
Helical wheel representation of the region
Lys through Ile
in the carboxyl terminus of
the AT
receptor. The wheel was constructed assuming a 100
degree turn/residue, based on the prediction that this region is
-helical. Note that the helix can be divided into predominately
basic and hydrophobic aspects, and the close proximity of Leu
and Tyr
(boxed) on the hydrophobic side.
Amino acids are represented by the single-letter code (K = Lys, Q = Gln, L = Leu, Y = Tyr, F = Phe, and I = Ile).
While a
helical prediction for this region of the AT receptor
contradicts the current dogma of a tyrosine-based
-turn as the
internalization motif, such a possibility is supported by the recent
NMR data of Wilde et al.(49) . These authors
synthesized a 21-amino-acid peptide corresponding to the carboxyl
terminus of TGN38, a type I integral membrane protein that recycles
between the plasma membrane and the trans-Golgi network. This
peptide which contained the sequence YQRL analogous to the common
internalization motif YXXhyro (hydro = hydrophobic
amino acid) displayed a NMR spectra consistent with a helical, not
-turn, conformation. Regardless of secondary structure, the region
corresponding to the terminal end (Phe
-Pro
)
of this putative AT
receptor helix, as noted by Hjorth et al. ( Fig. 3of 50), is preserved across species and
among subtypes of AT
receptors, which is indicative of
conserved function. Based on our mutational data and given that this
region is not conserved in the noninternalizing AT
receptor(23) , the function of this region may be
internalization.
Recent studies demonstrating the association of
epidermal growth factor receptors with the adaptor complex AP2 of the
endocytotic machinery (10, 12) , and another using
kinase-deficient epidermal growth factor receptors(11) , have
provided evidence that receptor endocytotic motifs are masked in the
absence of ligand binding. Occupancy of receptors by ligand promotes an
allosteric change which favors exposure of internalization motifs to
the endocytotic apparatus and also causes autophosphorylation which
amplifies the conformational changes and internalization. Although
GPCRs lack intrinsic kinase activity, many are rapidly phosphorylated
by cellular kinases on serine, threonine, and tyrosine residues in
response to agonist(51) , and there is evidence suggesting a
role for phosphorylation in endocytosis. For example, mutation of
serine/threonine residues in the -adrenergic receptor
inhibits internalization(52) , the serine/threonine region of
the gastrin releasing peptide receptor is required for
endocytosis(53) , and phosphorylation of M2 receptors by the
specific receptor kinase GRK2 coincides with increased rates of
internalization(54) . Thus, in the general context of an
internalization mechanism, phosphorylation of GPCRs appears to augment
endocytosis. One interpretation of our data, which identified two
carboxyl-terminal regions (one hydrophobic and one
serine/threonine-rich) important for AT
receptor
endocytosis, could be that upon AII binding a conformation change
occurs which leads to exposure of the hydrophobic residues and
initiates internalization. In addition, this conformational change
presumably promotes phosphorylation of the distal phosphate acceptor
residues which may maintain this exposed state. Conversely, an
allosteric change in this putative helical region may be required for
phosphorylation of distant sites, permitting the phosphorylated
residues to interact directly with the endocytotic machinery. However,
the propensity of hydrophobic and tyrosine residues in the
internalization motifs of many receptors predicts that the former
possibility is more likely.
In summary, we have identified two
distinct regions of the AT receptor necessary for the
efficient endocytosis of AII
AT
receptor complexes
and demonstrated a central role for Leu
and Tyr
in this process. These residues are closely aligned on the
hydrophobic face of a putative
-helix. Future studies will focus
on identifying a role for AT
receptor phosphorylation in
internalization and the possible interaction of the AT
receptor with the adaptor complexes that initiate the clustering
and endocytosis process. The truncated, deleted, and point-mutated
AT
receptors described in this study should prove useful
in delineating these processes for the AT
receptor and may
provide a more general understanding of the process of endocytosis for
other GPCRs.