(Received for publication, December 18, 1995; and in revised form, February 12, 1996)
From the
The seven transmembrane segments (TMs) of many G-protein-coupled receptors (GPCRs) are thought to form a cavity into which cognate ligands insert, leading to receptor activation. Residues lining the cavity are often essential for optimal ligand binding and/or signal transduction. The present studies evaluated whether residues lining the cavity also contribute to specificity, using GPCRs for the polypeptides parathyroid hormone (PTH) and secretin as models. These ligands display no sequence homology with one another, and neither ligand cross-reacts with the other's receptor. However, mutation of a single amino acid in the second TM of the secretin receptor to the corresponding residue in the PTH receptor (N192I) resulted in a receptor that binds and signals in response to PTH. The reciprocal mutation in the PTH receptor (I234N) likewise unmasked responsiveness to secretin. Neither mutation significantly altered the response of the receptors to their own ligands. The results suggest a model of specificity wherein TM residues near the extracellular surface of the receptor function as a selectivity filter that restricts access of inappropriate ligands to an activation site in the transmembrane cavity.
The superfamily of G-protein-coupled receptors (GPCRs) ()initiates the biological effects of a remarkably diverse
array of agonists, ranging from ions to glycoproteins(1) . Each
GPCR has encoded within it the structural information required for
appropriate ligand affinity and selectivity. Although all GPCRs share
the common topologic feature of seven transmembrane domains (TMs),
subfamilies of GPCRs differ markedly in the strategies used to maintain
ligand affinity and selectivity. At one extreme, determinants of
affinity and selectivity may reside exclusively in the TMs (e.g. opsin and adrenergic receptor
subfamilies)(2, 3) . At the other extreme, agonist
affinity and selectivity may be largely a function of residues in the
receptors' N-terminal extracellular domains (e.g. luteinizing hormone/thyrotropin receptor subfamily)(4) .
The present studies were designed to identify determinants of ligand
specificity in a recently recognized subfamily of GPCRs that includes
receptors for the peptide hormones secretin, parathyroid hormone
(PTH)/PTH-related protein (PTHrP), gastric inhibitory polypeptide,
calcitonin, and several others(5) . Receptors in this subfamily
resemble the larger superfamily members in displaying an apparent 7-TM
topology but do not share significant sequence homology with them.
Agonist binding to these receptors leads to activation of adenylyl
cyclase (via G) and, in several cases, phospholipase C (via
G
or a related G-protein). Although receptors in this
subfamily display 30-65% sequence homology with one another,
their cognate peptide ligands are structurally diverse. Thus, these
receptors are ideally suited for studies of receptor specificity using
molecular chimeras. We chose to initiate such studies with the
receptors for PTH/PTHrP and secretin, since their ligands are highly
divergent and accordingly do not cross-react with each other's
receptor.
Previous studies have suggested an important role for the relatively large N-terminal extracellular domain of PTH/PTHrP and secretin receptors in ligand binding affinity and specificity(6, 7, 8, 9) . However, the results of the present study point to a critical role of specific residues in the second TM domain of these receptors in maintaining ligand selectivity.
Molecular chimeras of PTH/PTHrP and secretin receptors
transiently expressed in COS-7 cells were utilized to assess the
contribution of the N-terminal extracellular domains in determining the
specificity of ligand binding and activation (cAMP production). As
expected, the Wt PTH/PTHrP receptor did not display specific binding of I-secretin or respond to secretin with an increase in
cAMP accumulation; likewise, the Wt secretin receptor did not bind
I-PTHrP-(1-34) or respond to bPTH-(1-34) with
an increase in cAMP (Fig. 1A). PTH/PTHrP receptors
containing the N-terminal extracellular domain of the secretin receptor
(PTH(SecNt)R) failed to bind or respond to secretin but displayed
significant specific binding of
I-PTHrP-(1-34) and
responded to bPTH-(1-34) with increased cAMP accumulation; the
reciprocal chimeric molecule, Sec(PTHNt)R, likewise failed to recognize
PTH/PTHrP receptor agonists but responded to secretin in assays of both
ligand binding and cAMP production (Fig. 1B). Each
chimeric receptor displayed functional activity that was reduced
compared with the Wt receptors. Thus, cAMP responses of PTH(SecNt)R and
Sec(PTHNt)R to maximally effective concentrations of ligand were 16 and
35% those of the Wt PTH and secretin receptors, respectively. Moreover,
the apparent affinities of the chimeric receptors were reduced about
300-fold by introduction of the heterologous N-terminal extracellular
domains (Fig. 1C), and this was associated with shifts
to the right in the dose-response curves for ligand-stimulated cAMP
production (EC
values of 100 nM for each chimeric
receptor compared with 3 nM for each Wt receptor). These
results are consistent with previous studies indicating that the
N-terminal extracellular domains of the PTH/PTHrP and secretin
receptors contribute to optimal binding
affinity(6, 7, 8, 9) ; they also
imply that determinants beyond the N-terminal extracellular domain must
play an important role in maintaining ligand specificity.
Figure 1:
A, PTH and secretin do not interact
with each other's receptor. Constructs encoding the Wt opossum
PTH/PTHrP receptor (PTHR) or Wt rat secretin receptor (SecR) were transiently transfected into COS-7 cells, and
functional activities were evaluated 3 days later. Left panel,
specific binding of I-PTHrP-(1-34) and
I-secretin (Sec). Right panel, cAMP
responses to maximal concentrations (5 µM) of
bPTH-(1-34) or secretin. Values are the mean ± S.E. of 10
experiments, each performed in triplicate. B, functional
properties of N-terminal PTH/secretin receptor chimeras. Sequence
homology between these receptors allowed us to exchange their
N-terminal extracellular domains by splicing their cDNAs at a position
encoding the extracellular end of the first transmembrane segment. This
yielded PTH/PTHrP receptors containing the N-terminal extracellular
domain of the secretin receptor (PTH(SecNt)R) and secretin
receptors containing the N-terminal extracellular domain of the
PTH/PTHrP receptor (Sec(PTHNt)R). Left panel,
specific binding of
I-PTHrP-(1-34) and
I-secretin. Right panel, cAMP responses to
maximal concentrations (5 µM) of bPTH-(1-34) or
secretin, expressed as a percent of the maximal response of the
corresponding Wt receptor. Values are the mean ± S.E. of three
experiments, each performed in triplicate. C, binding
affinities of Wt and chimeric receptors. Left panel,
competitive binding of
I-PTHrP-(1-34) to the Wt
PTH/PTHrP receptor (PTHR) and to the PTH(SecNt)R chimera in
the presence of increasing concentrations of unlabeled
PTH-(1-34). IC
values for PTH-(1-34) binding
were 3.2 nM and 1.4 µM for PTH/PTHrP receptor and
PTH(SecNt)R, respectively. Initial binding (no unlabeled PTH) was 11.2
and 2.1% of added
I-PTHrP-(1-34) for PTH/PTHrP
receptor and PTH(SecNt)R, respectively. Right panel,
competitive binding of
I-secretin to the Wt secretin
receptor (SecR) and to the Sec(PTHNt)R chimera in the presence
of increasing concentrations of unlabeled secretin. IC
values for secretin binding were 6.3 nM and 1.8
µM for secretin receptor and Sec(PTHNt)R, respectively.
Initial binding (no unlabeled secretin) was 12.3 and 3.3% of added
I-secretin for SecR and Sec(PTHNt)R,
respectively.
Recently,
we have shown that an arginine residue that is conserved in the
putative second membrane-spanning region (TMII) of the PTH/PTHrP and
secretin receptors is crucial for full agonist binding affinity and for
consequent receptor activation(16) . To determine whether
residues in TMII that differ between PTH/PTHrP and secretin receptors
contribute to ligand specificity, we evaluated the functional
properties of chimeric receptors in which the TMII domains were
exchanged. A secretin receptor bearing TMII of the PTH/PTHrP receptor
(Sec(PTHII)R) resembled the Wt secretin receptor in its affinity for
secretin; remarkably, this chimeric receptor (unlike the Wt secretin
receptor) also displayed specific binding of I-PTHrP-(1-34) (Fig. 2A).
Corresponding results were obtained with the reciprocal chimeric
PTH/PTHrP receptor (PTH(SecII)R), which bound PTH with an affinity
similar to that of the Wt PTH/PTHrP receptor but also displayed
specific binding of
I-secretin (Fig. 2B).
The affinities of these chimeric receptors for the heterologous ligands
were reduced about 40-60-fold compared with the Wt receptors,
consistent with a contribution of regions outside of TMII to optimal
binding affinity.
Figure 2:
Ligand binding to TMII chimeras of
PTH/secretin receptors. A, binding properties of secretin
receptors containing TMII of the PTH/PTHrP receptor (Sec(PTHII)R). Left panel, competitive binding of I-secretin to the Wt secretin receptor (SecR)
and to Sec(PTHII)R in the presence of increasing concentrations of
unlabeled secretin. Initial binding was 11.9 and 9.6% of added
radioligand for secretin receptor and Sec(PTHII)R, respectively. Right panel, competitive binding of
I-PTHrP-(1-34) to the Wt PTH/PTHrP receptor (PTHR) and to Sec(PTHII)R in the presence of increasing
concentrations of unlabeled PTH-(1-34). Initial binding was 10.4
and 3.9% of added radioligand for PTH/PTHrP receptor and Sec(PTHII)R,
respectively. B, binding properties of PTH/PTHrP receptors
containing TMII of the secretin receptor (PTH(SecII)R). Left panel, competitive binding of
I-PTHrP-(1-34) to the Wt PTH/PTHrP receptor and to
PTH(SecII)R in the presence of increasing concentrations of unlabeled
PTH-(1-34). Initial binding was 10.0 and 8.7% of added
radioligand for PTH/PTHrP receptor and PTH(SecII)R, respectively. Right panel, competitive binding of
I-secretin
to the Wt secretin receptor and to PTH(SecII)R in the presence of
increasing concentrations of unlabeled secretin. Initial binding was
11.6 and 1.9% of added radioligand for secretin receptor and
PTH(SecII)R, respectively. C, PTH and secretin (Sec)
compete for a common binding site in the TMII receptor chimeras. Left panel,
I-PTHrP binding to Sec(PTHII)R in
the presence of increasing concentrations of unlabeled
bPTH-(1-34) or secretin. Specific
I-PTHrP binding
was 4.58 ± 0.25% of added tracer (n = 9). Right panel,
I-secretin binding to PTH(SecII)R
in the presence of increasing concentrations of unlabeled
bPTH-(1-34) or secretin. Specific
I-secretin
binding was 2.6 ± 0.22% of added tracer (n = 9). D, signaling (cAMP) responses of COS-7 cells expressing
WtPTHR, WtSecR, PTH(SecII)R, and Sec(PTHII)R. Left panel,
response to secretin; right panel, response to
bPTH-(1-34).
These results demonstrate that the TMII PTH/secretin receptor chimeras are able to interact both with secretin and with PTH/PTHrP. Secretin does not share any amino acid sequence homology with either PTH or PTHrP, and it was thus of interest to determine whether the chimeras contained distinct binding sites for these ligands. Strikingly, PTH and secretin were roughly equipotent in the competitive inhibition of radioligand binding (Fig. 2C), indicating that the two ligands interact with similar affinities to a common binding site in the chimeric receptors.
The altered specificity of the chimeric receptors also extended to ligand-dependent signaling. Thus, Sec(PTHII)R signaled in response to secretin in a similar fashion to the Wt secretin receptor but also signaled in response to PTH; and PTH(SecII)R displayed a Wt signaling response to PTH but also signaled (albeit weakly) in response to secretin (Fig. 2D). Two other peptides whose receptors are members of the PTH/secretin receptor subfamily, gastric inhibitory polypeptide and calcitonin, failed to raise cAMP levels or to bind to COS-7 cells expressing the chimeric receptors (not shown).
Inspection of the sequences of TMII of the PTH/PTHrP and secretin receptors reveals that they are identical save for three positions; Ile-186, Leu-190, and Asn-192 in the secretin receptor correspond to Met-228, Val-232, and Ile-234 in the PTH/PTHrP receptor (Fig. 3). Asn-192 (secretin receptor)/Ile-234 (PTH/PTHrP receptor) is predicted to face the TM cavity, lining the polar face of TMII, immediately adjacent to a serine residue and one helical turn on the extracellular side of an arginine residue, shown to be essential for optimal binding affinity and efficient signaling to adenylyl cyclase(16) . Mutation of N192I in the secretin receptor reproduced the specificity change for ligand binding (Fig. 4A) and cAMP response (Fig. 4B) seen with the TMII chimera bearing mutations at all three positions, whereas (I186M, L190V)-secretin receptors failed to respond to PTH (not shown). Conversely, (I234N)-PTH/PTHrP receptors displayed a Wt response to PTH and also responded to secretin (Fig. 4). The (N192I)-secretin receptor and (I234N)PTH/PTHrP receptor exhibited dose-response curves for ligand binding and cAMP production that were indistinguishable from those of Sec(PTHII)R and PTH(SecII)R, respectively (not shown). Thus, in the Wt secretin and PTH/PTHrP receptors, Asn-192 and Ile-234 may confer specificity by preventing the non-cognate ligands from making contact with key TM residues. This restrictive function is partially abrogated in the (N192I)-secretin and (I234N)-PTH/PTHrP receptors, allowing either ligand to contact the activation site and thus to trigger signaling.
Figure 3: Schematic showing the sequences within the putative second transmembrane regions of the PTH/PTHrP receptor (PTHR, left) and secretin receptor (SECR, right). Differing amino acids, the targets of mutagenesis in the present studies, are highlighted. Those residues predicted by helical wheel analysis to be aligned on the polar face of TM II, facing the TM cavity, are shown along the right edge of each diagram. The extracellular (top) and intracellular (bottom) boundaries of the TM domains are based on sequence alignment of members of the PTH/secretin receptor subfamily of GPCRs.
Figure 4:
A, a
point mutation (N192I) in the secretin receptor reproduces the altered
specificity of the Sec(PTHII)R chimera. Left panel, specific
binding of I-PTHrP-(1-34) to the Wt secretin
receptor, to Sec(PTHII)R, and to (N192I)-SecR. Right panel,
cAMP response to 5 µM bPTH-(1-34) of COS-7 cells
expressing these receptors. Values are the mean ± S.E. of three
experiments, each performed in triplicate. B, a point mutation
(I234N) in the PTH/PTHrP receptor reproduces the altered specificity of
the PTH(SecII)R chimera. Left panel, specific binding of
I-secretin to the Wt PTH/PTHrP receptor, to PTH(SecII)R,
and to (I234N)PTH/PTHrP receptor. Right panel, cAMP response
to 5 µM secretin of COS-7 cells expressing these
receptors. Values are the mean ± S.E. of three experiments, each
performed in triplicate.
The receptors for PTH/PTHrP and secretin are members of a subfamily of G-protein-coupled receptors, which display sequence homologies not evident in other superfamily members(5) . They presumably arose by divergence from a common ancestral receptor and have retained very similar modes of signal transduction but have evolved responsiveness to diverse peptide ligands. The present results provide a new perspective on the possible nature of this diversification. Receptors in this subfamily may be modular in nature, with activation requiring an interaction of the peptide ligand with a site within the TM domains, as has been suggested for GPCRs for other peptides(17, 18, 19, 20, 21, 22) . The activation site appears to recognize features common even to non-homologous peptide ligands such as PTH and secretin, and therefore this site is unlikely to provide the major elements of receptor specificity. In this view, specificity is imposed (at least in part) by TM residues that face the cavity and restrict access of the ligand to the activation site. These residues would serve a function analogous to that of the selectivity filter in ion channels(23, 24) . Indeed, point mutations of residues comprising ion channel selectivity filters are also known to produce dramatic changes in selectivity(25, 26, 27, 28) . The use of a selectivity filter to establish specificity in a peptide receptor subfamily might allow relatively subtle evolutionary modifications to alter the receptor's ligand specificity without compromising the function of its signal-transducing elements.
In summary, results of the present study indicate that discrete, specialized components in the TM domain of the G-protein-coupled receptors for PTH/PTHrP and secretin function to limit access of peptide ligands to the active site and thereby to impose specificity. This function can, in part, be abrogated by targeted point mutations of these residues. It will be of considerable interest to determine the physicochemical basis of this restriction and whether similar mechanisms are operative in other G-protein-coupled receptors for peptide ligands.