(Received for publication, November 27, 1995; and in revised form, December 11, 1995)
From the
G protein-coupled receptors (GPCRs) have seven hydrophobic
domains, which are thought to span the lipid bilayer as helical
transmembrane domains (TMDs). The tertiary structure of GPCRs has not
been determined; however, molecular models of GPCRs have generally been
based on bacteriorhodopsin, which is functionally unrelated to GPCRs
but has a similar secondary structure. We sought to examine the
validity of using bacteriorhodopsin as a scaffold for GPCR model
building by experimentally determining the orientation of the TMDs of
adrenergic receptors in the plasma membrane. In separate experiments,
three sequential amino acid residues (Leu-310, Leu-311, Asn-312) in TMD
VII of the
adrenoreceptor were mutated to the amino
acids found in the homologous domain of the
adrenoreceptor (Phe, Phe, Phe). Exchange of Asn-312 and Leu-311
in the
adrenoreceptor resulted in nonfunctional
proteins, most likely due to incompatibility of the introduced bulky
phenylalanine side chain with adjacent structural domains in the
adrenoreceptor. This structural incompatibility was
``repaired'' by replacing the specific
TMD
sequence with an
receptor sequence. TMD I and TMD II
complemented the Asn-312
Phe mutation, and TMD III and TMD VI
complemented the Leu-311
Phe mutation. These results indicate
that TMDs I, II, III, and VI surround TMD VII in a counterclockwise
orientation analogous to the orientation of TMDs in bacteriorhodopsin.
Adrenergic receptors are one of the best characterized class of
the family of G protein-coupled receptors for hormones and
neurotransmitters. G protein-coupled receptors exhibit a common
secondary structure consisting of seven hydrophobic segments that are
thought to represent helical membrane-spanning domains. In this
paper the hydrophobic segments will be referred to as transmembrane
domains (TMD). (
)A high resolution three-dimensional
structure of adrenergic and other G protein-coupled receptors is
lacking because of the difficulties inherent in producing, purifying,
and crystallizing integral membrane proteins. Without these data,
investigators have used the structural information from an analogous
protein, bacteriorhodopsin, as a template from which to generate
molecular models of adrenoreceptors(1) . The rationale for
using bacteriorhodopsin, a prokaryotic proton pump, as a
``structural scaffold'' for human adrenoreceptors is based on
the seven-transmembrane
helices in bacteriorhodopsin, which are
thought to correspond topographically to the seven hydrophobic domains
present in G protein-coupled receptors. But is bacteriorhodopsin
structurally similar enough to employ as a model for G protein-coupled
receptors? While bacteriorhodopsin does share with rhodopsin (a G
protein-coupled receptor) the photoisomerization of the retinal
chromophore, it has less than 10% sequence homology with G
protein-coupled receptors and is not coupled to G proteins. When
bacteriorhodopsin and rhodopsin are compared at a low resolution (9
Å) the projection footprints of the helical arrangements are
different(2, 3) .
Because of the lack of
independent evidence that hormone and neurotransmitter G
protein-coupled receptors are structurally analogous to
bacteriorhodopsin, we sought to identify whether the membrane-spanning
helices of adrenoreceptors were arranged clockwise as depicted in
several models (4, 5) or counterclockwise (6) (as for bacteriorhodopsin) when viewed from the outside of
the cell. We have used a strategy involving
/
adrenoreceptor chimeric receptors
to identify intramolecular interactions between specific amino acids on
TMD VII and other TMDs. Our results provide evidence that
adrenoreceptors exhibit the same helical orientation known to be
present in bacteriorhodopsin.
The construction and functional analysis of chimeric
receptors from and
adrenoreceptors
have provided useful information about adrenoreceptor
structure(10, 11, 12, 13) . These
two receptors are both activated by the catecholamine epinephrine but
can be readily distinguished pharmacologically using subtype-selective
agonists and antagonists. The
adrenoreceptor couples
to G
and thereby inhibits adenylyl cyclase while the
adrenoreceptor couples to G
and activates
adenylyl cyclase. The binding specificity of wild-type
adrenoreceptor can be converted to that of a
adrenoreceptor by mutating Phe-412
Asn in the seventh
transmembrane domain(11) ; however, the ``mirror
image'' mutation (Asn-312
Phe on the
adrenoreceptor) is nonfunctional requiring the additional
substitution of TMDs I and II from the
adrenoreceptor
(see CRS11 in Fig. 1) to restore function(12) .
CRS 11 has
adrenoreceptor domains essential for
coupling to G
, and binding of epinephrine to CRS11 leads to
activation of adenylyl cyclase (Fig. 1). Pharmacologically CRS11
behaves more like an
adrenoreceptor(12, 13) . Thus the more bulky
phenylalanine residue at position 312 in TMD VII requires accommodation
by complementary changes in TMDs I and II, suggesting that the amino
acid at position 312 in the
adrenoreceptor faces TMDs
I or I and II. These results are in agreement with more recent studies
on the 5HT
receptor demonstrating a specific interaction
between TMD II and TMD VII(14) . We originally depicted the
interaction between TMD VII and TMDs I and II in a model where the TMDs
were arranged in a clockwise orientation(12) ; however,
orientation of the helices in either a counterclockwise (Fig. 2A) or clockwise (Fig. 2B) manner
is consistent with these data.
Figure 1:
Functional properties of chimeric
receptors. Receptors were transfected into COS-7 cells as described
under ``Experimental Procedures.'' The adrenoreceptor sequence inserted into the
adrenoreceptor to make each chimer is indicated (TM,
transmembrane domain; AA, amino acid residue) and is shown as black-filled in the receptor diagrams. Transfection
efficiency, quantified by immunocytochemistry, varied between 20 and
40%. Both the radiolabeled ligand binding assays (to determine the K
values) and the adenylyl cyclase assays
(to determine the EC
values) were performed at least three
times, each in triplicate. Maximal epinephrine-induced increase in
adenylyl cyclase stimulation (maximal/basal) for functional receptors
was as follows: 1.76 ± 0.20 for CRS11, 1.51 ± 0.08 for
CRS121, and 2.40 ± 0.18 for CRS117. All of the receptor chimeras
contain sequences previously shown to be required for G
activation (10) and for binding to
antagonists such as atipamezole(12, 13) .
Epinephrine is an agonist for both
and
adrenoreceptors.
Figure 2:
The arrangement of the transmembrane
domains (I-VII) as viewed from the outside of the cell.
domains are shown as black circles with white letters, and
domains are shown as open circles with black letters. The replacement of
asparagine 312 in the
transmembrane domain VII by
phenylalanine (F) requires exchange of transmembrane domains I
and II with the
adrenoreceptor sequence to be
functional(12) . This suggests that TMDs I and II are adjacent
to position 312 in TMD VII, and this can be accommodated by either a
counterclockwise (A) or a clockwise (B) orientation.
Substitution with the
residue phenylalanine (F) for leucine (L) at position 311 in the
transmembrane domain VII requires
transmembrane domains III and VI to be functional. This suggests
that TMDs III and VI are adjacent to position 311 in TMD VII. The
findings are most easily explained by a counterclockwise (C)
rather than a clockwise (D) orientation of TMDs I, II, III,
and VI around TMD VII.
To determine which of these two
orientations pertained, we examined the effect of mutations at
positions 311 and 310. The orientation of positions 311 and 310 shown
in Fig. 2is based on the assumption that TMD VII traverses the
membrane as a right-handed helix. Leu-311 was mutated to Phe (the
amino acid in the homologous position of the
adrenoreceptor) to determine if complementary changes were
required in the neighboring TMDs of this novel receptor (CRS112) to
maintain receptor function. Immunocytochemical studies confirmed that
CRS112 was expressed in the plasma membrane of the transfected COS-7
cells (data not shown), but membranes prepared from these receptors
displayed neither
nor
antagonist
binding, and adenylyl cyclase activity could not be stimulated with
epinephrine (Fig. 1) even though this receptor retains sequences
required for G
coupling(10) . If adrenoreceptor
TMDs have an arrangement similar to bacteriorhodopsin, residue 311
should face in the direction of other TMDs, most likely TMDs III and/or
VI (Fig. 2A). Therefore, new receptor constructs were
made in which either TMD III (CRS 113) or TMD VI (CRS 114) of CRS 112
was replaced by its
homolog. Again, both these new
mutant receptors were appropriately expressed in plasma membranes of
mammalian cells (data not shown) but displayed neither adrenergic
radiolabeled ligand binding nor adenylyl cyclase stimulation (Fig. 1). However, when TMDs III and VI of CRS112 were both
replaced by their
homologs, epinephrine-stimulated
adenylyl cyclase activity and radiolabeled ligand binding were fully
recovered in this mutant (CRS121) and were similar to CRS11 (Fig. 1). These data are most consistent with a counterclockwise
orientation of TMDs as shown in Fig. 2C and appear to
be incompatible with a clockwise helical orientation (Fig. 2D) since the short hydrophilic sequence between
TMDs II and III would not likely accommodate their separation by TMD
VII.
According to the arrangement of TMDs in Fig. 2C, amino acid 310 (rotated a further 100°
counterclockwise from 311) should not interact with neighboring TMDs.
If this prediction is correct, then mutating Leu-310 in CRS11 to Phe
(found in the homologous position of the adrenoreceptor) should not affect agonist activation or binding
function. This prediction was borne out since CRS117 (Fig. 1)
has similar epinephrine-stimulated adenylyl cyclase activity and
adrenergic ligand binding to that of CRS11.
A high resolution
tertiary structure of G protein-coupled receptors has not yet been
determined because of the difficulty in extracting large amounts of
pure protein from the natural membranes for crystallographic studies.
Cryoelectron microscopy of two-dimensional crystals has been used to
study the structure of rhodopsin(2, 3) ; however, the
resolution (9 Å) of the structure obtained from these
studies is not yet sufficient to determine the arrangement of the TMDs.
In the absence of high resolution biophysical data, structural models
have been devised, based largely on the folding pattern of
bacteriorhodopsin. The data presented here provide evidence for
specific intramolecular interactions between position 311 in TMD VII of
the
adrenoreceptor and TMDs III and VI and, together
with our previous results(12) , support the hypothesis that the
human adrenoreceptor exhibits the same helical orientation that is
known to be present in bacteriorhodopsin(15) . This structural
similarity increases the likelihood that bacteriorhodopsin is an
appropriate scaffold on which to ``drape'' the adrenoreceptor
sequence and provides the experimental underpinnings for rational drug
design using a molecular model of adrenergic receptors based on
bacteriorhodopsin.