(Received for publication, October 24, 1994)
From the
We have examined the role of transmembrane domain amino acids in
conferring subtype-selective ligand affinity to the human
cholecystokinin-B (CCK-B)/gastrin receptor. Fifty-eight residues were
sequentially replaced by the corresponding amino acids from the
pharmacologically distinct CCK-A receptor subtype. I-CCK-8 competition binding experiments were performed to
compare all mutant CCK-B/gastrin receptor constructs with the wild type
control. Affinities for the nonselective agonist, CCK-8, as well as the
subtype-selective peptide (gastrin), peptide-derived (PD135,158), and
nonpeptide (L365,260 and L364,718) ligands were assessed. All of the
mutants retained relatively high affinity for CCK-8, suggesting that
the tertiary structure of these receptors was well maintained. Only
eight of the amino acid substitutions had a significant effect on
subtype selective binding. When compared with the wild type, single
point mutations in the CCK-B/gastrin receptor decreased affinity for
gastrin, L365,260, and PD135,158 up to 17-, 23-, and 61-fold,
respectively. In contrast, the affinity for L364,718 increased up to
63-fold. None of the single amino acid substitutions, however, was
sufficient to fully account for the subtype selectivity of any tested
compound. Rather, CCK-B/gastrin receptor affinity appears to be
influenced by multiple residues acting in concert. The 8
pharmacologically important amino acids cluster in the portion of the
transmembrane domains adjacent to the cell surface. The spatial
orientation of these residues was analyzed with a rhodopsin-based
three-dimensional model of G-protein coupled receptor structure
(Baldwin, J. M.(1993) EMBO J. 12, 1693-1703). This model
[Abstract]
predicts that the 8 crucial residues project into a putative ligand
pocket, similar to the one which is well established for biogenic amine
receptors (Caron, M. G., and Lefkowitz, R. J.(1993) Recent Prog.
Horm. Res. 48, 277-290; Strader, C. D., Sigal, I. S., and
Dixon, R. A.(1989) Trends Pharmacol. Sci. 10, Dec. Suppl.,
26-30).
Cholecystokinin octapeptide (CCK-8) ()and gastrin are
structurally related regulatory peptides which share high affinity for
the CCK-B/gastrin receptor. This receptor is widely expressed in the
central nervous system, as well as in a number of peripheral tissues
including smooth muscle, pancreas, and stomach. In the central nervous
system, the CCK-B/gastrin receptor has been implicated in anxiety,
panic attacks, and the perception of pain (4, 5, 6, 7, 8) , making it
a potentially important clinical target for receptor-specific
antagonists. The CCK-B/gastrin receptor in the stomach has also been
extensively studied and has been shown to modulate acid secretion as
well as the proliferation of enterochromaffin-like cells(9) .
The CCK-B/gastrin receptor is a member of the seven-transmembrane
domain -adrenergic receptor superfamily. Within this broad group,
it is well established that the transmembrane domain residues of
biogenic amine receptors are the primary determinants of ligand
affinity(2, 3) . In contrast, relatively little is
known about the molecular basis for ligand affinity to peptide
receptors. We have previously shown that differences in the affinities
for nonpeptide antagonists between the human and canine CCK-B/gastrin
receptors result from interspecies variation in a single transmembrane
domain amino acid(10) . In addition, the potential importance
of the peptide receptor transmembrane domains in determining ligand
affinity has been further illustrated by mutational analysis of the
neurokinin(11, 12, 13, 14, 15, 16) and
angiotensin (17) receptors.
The aim of this study was to analyze the role of CCK-B/gastrin receptor transmembrane domain amino acids in determining high affinity binding of subtype-selective agonists and antagonists. The strategy we utilized to identify potentially important residues was to compare the human CCK-B/gastrin receptor with the other known CCK receptor subtype, CCK-A. The two subtypes share 47% amino acid identity, as well as high affinity for CCK-8. Pharmacologically, the CCK-B/gastrin receptor can be distinguished from the CCK-A subtype by its unique affinity profile for peptide as well as nonpeptide ligands(9) . Comparison between the transmembrane segment amino acid sequences of the CCK-B/gastrin and the CCK-A receptors revealed 58 differences, residues which could potentially confer affinities characteristic of a CCK-B/gastrin receptor. These divergent amino acids were sequentially substituted in the CCK-B/gastrin receptor with their CCK-A homologs. The resultant mutant receptors were assessed for alteration in their agonist and antagonist binding profiles. We report here that 8 of the 58 residue substitutions significantly influence ligand affinity for the CCK-B/gastrin receptor.
Figure 1:
Amino acid substitutions which alter
ligand affinity cluster in the outer third of the transmembrane domains
adjacent to the extracellular space. The inset shows a
schematic representation of the CCK-B/gastrin receptor; transmembrane
domains are numbered as in the main figure. The upper line shows the amino acid sequence corresponding to transmembrane
domains of the human CCK-B/gastrin
receptor(32, 33, 34) . The middle line shows residues in the rat CCK-A receptor sequence (22) which differ from the human CCK-B/gastrin receptor. The lower line shows human CCK-A receptor sequence (23, 24) which diverges from the rat CCK-A receptor.
Divergent CCK-A receptor residues were sequentially substituted into
the CCK-B/gastrin receptor as units of 1-4 amino acids/mutant (underlined). Analysis of mutants was done by I-CCK-8 binding experiments, using as competitors two
agonists, sulfated CCK-8 and gastrin (B-specific), and two
benzodiazepine-based subtype-selective antagonists, L365,260
(B-specific) and L364,718 (A-specific), n
2. The wild
type receptor was tested in parallel for every experiment, and mutant
IC
values were always expressed relative to wild type.
Data shown for each mutant represent only the largest affinity shift
which was observed for any of the four ligands tested. Units on the y axis correspond to either mutant/wild type IC
ratios for affinity decreases or to wild type/mutant
IC
ratios for affinity increases. Table 1shows the complete binding profile of mutants with an
IC
ratio greater than 2.5 for any ligand. For 3 of the
amino acids in the CCK-B/gastrin receptor, two different mutants were
tested, one corresponding to rat and the other to human CCK-A sequence.
In these cases, a large dot denotes IC
ratios of
mutants which include human CCK-A receptor sequence. Amino acid
numbering corresponds to the position in the human CCK-B/gastrin
receptor. N, amino terminus; C, carboxyl terminus; EC, extracellular loop; IC, intracellular space; TM, transmembrane domain; aa, amino
acids.
Figure 3:
Overlapping cross-sectional views of the
seven transmembrane domains of the CCK-B/gastrin receptor. This diagram
is based on a model of G-protein coupled receptors (1) derived
from the two-dimensional crystalline structure of
rhodopsin(25) . Large circles represent -helices
1-7. The inward faces of the seven helices define the borders of
a putative ligand pocket. The small numbered circles correspond to amino acids in the human CCK-B/gastrin receptor. Gray shading indicates amino acid identity between the human
CCK-A and CCK-B/gastrin receptors. Open circles represent
pharmacologically silent differences, whereas black solid circles mark amino acids in the CCK-B/gastrin receptor which result in
affinity shifts when substituted with their CCK-A counterparts.
CCK-B/gastrin receptor residues are shown in single letter amino acid
code, followed by homologous CCK-A residues in parentheses. A, the outer third of the transmembrane domains adjacent to
the extracellular space defines a putative ``ligand binding
pocket.'' Pharmacologically significant amino acid substitutions
The transmembrane domain amino acid sequences of the human
CCK-B/gastrin and CCK-A receptors differ in 58 positions. For each
residue where the sequence of the two subtypes is not conserved,
CCK-B/gastrin receptor amino acids were substituted with the
corresponding CCK-A homolog. In total, 38 mutant receptors (1-4
amino acid substitutions/mutant) were constructed. Initially, the amino
acid substitutions were based on the rat CCK-A receptor
sequence(22) . When the human CCK-A receptor sequence became
available(23, 24) , several additional mutants were
constructed to include all human CCK-A residues which differ from the
rat (Fig. 1). Receptors were expressed in COS-7 cells, and the
affinities for CCK-8, gastrin, L365,260, and L364,718 were determined
by I-CCK-8 competition binding experiments. Only 8 of the
58 divergent amino acids, Arg
, Thr
,
Ser
, Ser
, Val
,
Tyr
, Thr
, and His
, shifted
affinity for any of the tested competitors by more than 2.5-fold
relative to wild type (p < 0.05) (Table 1).
All
mutant receptors except for H376L had IC values for CCK-8
in the range between the wild type CCK-B/gastrin and CCK-A receptors
(7.6-fold difference), suggesting that the tertiary structure of all
receptors was relatively well conserved. While the substitution mutant
H376L had a slight decrease in affinity for CCK-8 (to approximately
4-fold lower than observed for the CCK-A receptor), it also
demonstrated a 63-fold increase in affinity for the CCK-A receptor
specific antagonist, L364,718, in comparison with the wild type
CCK-B/gastrin receptor (Table 1). This suggests that the overall
conformation of the H376L mutant remained intact.
We have previously shown that V349I increased affinity for L364,718 by 6-fold relative to the wild type CCK-B/gastrin receptor(10) . A similar 5-fold increase in affinity was observed with Y350F (Table 1). These affinity shifts were additive; the double mutant V349I/Y350F increased affinity for L364,718 thirty-eight fold in comparison with wild type (Fig. 2). This combination mutant illustrates how multiple residues may interact to determine overall affinity for a given ligand. In addition, the V349I/Y350F substitution demonstrates how agonist and antagonist affinities can be independently affected, since this mutation did not alter affinity for either CCK-8 or gastrin.
Figure 2:
Multiple amino acids act in concert to
determine L364,718 affinity. Comparison of I-CCK-8
binding to wild type and mutant CCK-B/gastrin receptors in the presence
of increasing concentrations of L364,718. Respective IC
values (nM ± S.E.) for L364,718 were as follows:
wild type (WT), 204 ± 10 (n = 11);
V349I, 28.1 ± 3.7 (n = 3); Y350F, 31.9 ±
4.1 (n = 7); V349I/Y350F, 5.6 ± 0.5 (n = 5).
Four amino acid substitutions, R57Q, S131T, S219H, and H376L, decreased affinity for gastrin, an agonist with high specificity for the CCK-B/gastrin receptor subtype. In each of these mutant receptors, the loss of affinity for gastrin was mirrored by an increase in affinity for the CCK-A receptor specific antagonist, L364,718, by 6.7-, 3.6-, 4.8-, and 62.5-fold, respectively (Table 1). The increases in affinity for L364,718 suggest that these substituted receptors are not merely ``loss of function'' variants. Rather, these mutations in the CCK-B/gastrin receptor shift its ligand specificity toward that of the CCK-A subtype.
Four amino acids substitutions, T111N, S219H
or Q, and H376L, reduced affinity for the CCK-B/gastrin receptor
specific antagonist, L365,260 (Table 1). The substitution, T111N,
decreased L365,260 affinity with little effect on agonist binding, as
did substitution of Ser with the corresponding residue
(glutamine) from the rat CCK-A receptor. In contrast, S219H, the human
homolog of the same mutant, and H376L not only decreased L365,260
binding, but also reduced affinities for CCK-8 and gastrin closer to
the values expected for the CCK-A receptor.
Taken together, our data indicate that no single residue substitution is sufficient to completely reverse subtype selectivity for gastrin, L365,260, or L364,718. It therefore appears that ligand specificity is determined by multiple amino acids working in concert.
Hydropathy analysis of the CCK-B/gastrin receptor sequence suggests that the 8 amino acids which affect ligand affinity cluster toward the outer third of the transmembrane domains, adjacent to the extracellular space (Fig. 1). To further examine the potential spatial orientation of these CCK-B/gastrin receptor amino acids, we utilized a rhodopsin-based model of G-protein coupled receptor transmembrane domain structure(1, 25) . As shown in Fig. 3, the model depicts three overlapping transmembrane domain cross-sectional views, partitioned into an outer (A), middle (B), and inner third (C). When the CCK-B/gastrin receptor is examined using this model, all 8 amino acids which alter subtype selective binding are positioned on the inward pointing faces of the helices, outlining a putative ligand pocket within the CCK-B/gastrin receptor (Fig. 3A).
Neither the middle nor the inner thirds of the transmembrane segments appear to be important in determining subtype selective ligand affinity. In the middle third of the transmembrane domains, amino acids which project into the putative ``binding pocket'' tend to be identical when the CCK-B/gastrin and CCK-A receptors are compared (Fig. 3B). This suggests that the central portion of the transmembrane domain helices provides a conserved structural requirement for both CCK receptor subtypes. As in Fig. 3A, functionally silent amino acid substitutions tend to project away from the putative pocket. In the inner third of the transmembrane domains, none of the residue substitutions altered the affinity for subtype-selective compounds (Fig. 3C).
The large number (50) of pharmacologically silent amino acid substitutions makes it unlikely that the observed affinity shifts result from random disruption of transmembrane domain helical packing. The specificity of the eight mutation-induced functional changes is also supported by the predictable configuration of these residues in the extracellular third of the membrane. The significance of this clustering is further highlighted by the relative absence of residues which determine affinity in the middle and intracellular cross-sections of the transmembrane domain helices (Fig. 3, B and C).
To further support the hypothesis that the amino acids
projecting into the ligand pocket affect subtype selectivity, we
examined the binding of PD135,158 (CAM-1028), which exemplifies an
additional class of CCK-B/gastrin receptor antagonists(26) .
This compound binds with much higher affinity to the wild type
CCK-B/gastrin than to the CCK-A receptor (IC ratio
= 250.5 ± 35.8; mean ± S.E. from three independent
experiments). Substitutions in three of the eight positions previously
shown to influence ligand binding, T111N, S219H, and T354A,
significantly decreased PD135,158 affinity toward the value expected
for a CCK-A receptor: 3.7 ± 0.4, 60.9 ± 20.1, and 5.3
± 0.7-fold, respectively (mean IC
ratio ±
S.E., n
2, p < 0.01).
Further studies are required to determine whether the CCK-B/gastrin receptor residues influencing affinity form a true ligand pocket or indirectly affect agonist and antagonist binding. Localization of these amino acids to the transmembrane domains is based on hydropathy algorithms and comparison with other G-protein-coupled receptors(1) . The distinction between extracellular loop amino acids which are close to the membrane, and residues which are within the outer portion of the transmembrane domains is a best approximation. Thus, some of the amino acids shown in Table 1may actually influence ligand affinity as part of an extracellular loop. The contribution of the extracellular loops to peptide receptor affinity is well supported by work on the neurokinin-1 (12, 27, 28) and thrombin (29) receptors. It is therefore likely that the complete CCK-B/gastrin receptor binding pocket is defined by both transmembrane domain and extracellular loop amino acids.
To investigate whether
the proposed transmembrane domain binding pocket is applicable to other
peptide receptors, the human CCK-B/gastrin and the neurokinin-1 (NK-1)
receptors were compared. Ten transmembrane domain residues in the human
NK-1 receptor, Asn, Asn
, Val
,
Gln
, Ser
, His
,
His
, Tyr
, Ile
, and
Met
, have been implicated in ligand affinity by
mutational
analysis(11, 12, 13, 14, 15, 16) .
When projected into the CCK-B/gastrin receptor model, all 10 NK-1
residues are found on the inward face of the transmembrane domain
helices and project toward the putative binding pocket (Fig. 3A). Four of these 10 residues map to the same
positions as amino acids which influence CCK-B/gastrin receptor subtype
selectivity, T111N, S219H, Y350F, and H376L. These observations suggest
that the binding pocket of the NK-1 and the CCK-B/gastrin receptors are
structurally similar.
The extent to which these findings can be generalized remains to be further investigated. Two established binding determinants in other peptide receptors, tyrosine 106 of the thyrotropin-releasing hormone receptor (30) and lysine 181 of the endothelin B receptor(31) , appear to project into the putative ligand pocket (Fig. 3A). In contrast, a number of affinity determinants in the angiotensin AT-1 receptor are not consistent with the rhodopsin-based model. Some of the crucial AT-1 receptor transmembrane domain amino acids project away from the binding pocket and others are located in close proximity to the cytoplasm(17) .
In summary, our analysis suggests that the specific ligand affinity profile of the CCK-B/gastrin receptor is, in part, determined by residues which occupy the outer third of the transmembrane domains adjacent to the extracellular space and project into a putative ligand pocket in the membrane. An analogous model is well established for the biogenic amine receptors (2, 3) , suggesting that the organization of the transmembrane domain binding determinants has been conserved, at least to some degree, across the broad family of G-protein-coupled receptors. These findings should enhance the understanding of high affinity ligand binding to the CCK-B/gastrin receptor, which may expedite the design and development of subtype selective receptor antagonists.