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School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom
Address all correspondence and requests for reprints to: Dr. Mark Wheatley, School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom. E-mail: m.wheatley{at}bham.ac.uk.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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It is of fundamental importance to define the ligand binding site within the receptor architecture and furthermore, to understand how agonist:receptor interactions differ from antagonist:receptor interactions. Defining the differences at the molecular level between the agonist:receptor complex and antagonist:receptor complex is central to our understanding of GPCR activation by hormones and to the rational design of receptor-specific agonists and antagonists. For biogenic amines, the ligand binding site is located in a hydrophobic binding pocket within the transmembrane domains (11, 12, 13). Binding of larger peptide hormones has been reported to involve additional extracellular domains (14, 15). Indeed, the very large glycohormones such as LH, will bind with high affinity to the isolated N-terminus of the receptor (16). For the V1aR, the AVP binding site has been proposed to be buried in a 15–20 Å deep cleft within the transmembrane domain (17). Peptide mimetic studies (18), photoaffinity labeling (19) and site-directed mutagenesis (20, 21) have also indicated the involvement of the first extracellular loop in ligand binding. In addition, it has been established recently that the N-terminus of the V1aR is required for binding agonists but not antagonists (22). In this study, we identify individual residues within the N-terminus that provided this agonistspecific interaction, evaluate the side-chain properties required for this role and suggest a plausible mechanism of action.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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2–47]V1aR), was decreased relative to wild-type V1aR by 2,000-fold. Furthermore, the importance of this domain was restricted to agonist binding, as its absence in the [2–47]V1aR construct did not influence the binding of cyclic antagonist, linear antagonist, or nonpeptide antagonist (22). The aim of this study was to identify individual residue(s) within the N-terminus of the V1aR that are responsible for this agonist-specific interaction.
Three truncated V1aR constructs were engineered to address which sections of the N-terminus contribute the agonist-specific binding epitopes. Truncations were made at Pro41, Asp44, and Asn47 as indicated in Fig. 1
, with the initiation methionine retained in each case. These constructs were termed [2–41]V1aR, [2–44]V1aR and [2–47]V1aR, respectively, where the numbering refers to the amino acid residues deleted. Receptor constructs were expressed in HEK 293T cells. The wild-type V1aR and truncated constructs were all expressed at the same level of approximately 1–2 pmol/mg protein. The pharmacological characteristics of the truncated receptor constructs [2–41]V1aR, [2–44]V1aR and [2–47]V1aR were determined and compared with wild-type V1aR. In each case, competition radioligand binding curves were determined using the natural agonist AVP (Fig. 2A) and three different structural classes of antagonist: 1) cyclic peptide antagonist [d(CH2)5Tyr(Me)2AVP (23)] containing a twenty-membered ring formed by a disulfide bond between Cys1 and Cys6; 2) linear peptide antagonist ([PhAcD-Tyr(Me)2Arg6Tyr(NH2)9]AVP) (24); and 3) nonpeptide antagonist [SR 49059; (25)]. The Kd values are presented in Table 1, corrected for radioligand occupancy.
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2–41]V1aR) had no effect on the binding of the agonist AVP or the three different classes of antagonist (Fig. 2A
and Table 1). Further truncation of the N-terminus to Asp44 (construct [2–44]V1aR) resulted in a slight decrease in AVP affinity with the Kd increasing approximately 6-fold (Fig. 2A and Table 1). In contrast, the binding affinities for all three different classes of antagonist remained unchanged from wild-type receptor values. Truncation of the N-terminus as far as Asn47 (construct [2–47]V1aR) resulted in a 2,000-fold decrease in affinity for AVP but did not affect the binding of antagonists (Fig. 2A and Table 1). It could be concluded from these findings, that the region of the N-terminus required for high affinity agonist binding comprised six residues from Leu42 to Asn47.
Identification of an N-Terminal Patch of Residues Contributing to High Affinity Agonist Binding
To identify the contribution to agonist binding provided by the individual residues present in the subdomain, the residues Leu42 to Asn47 inclusive were mutated individually to alanine (Fig. 1). The pharmacological characteristics of each of these alanylsubstituted receptors were investigated and compared with wild-type V1aR. Competition radioligand binding curves were determined for AVP (Fig. 2, B and C) and for the three different classes of antagonist. The Kd values for AVP and the antagonists are presented in Table 1, corrected for radioligand occupancy. The wild-type V1aR and mutant receptors were all expressed at the same level of approximately 1–2 pmol/mg protein.
The mutant constructs [L42A]V1aR, [G43A]V1aR and [D44A]V1aR exhibited a pharmacological profile similar to wild-type receptor (Fig. 2B and Table 1). Individually, these residues do not appear to be involved in agonist recognition. However, characterization of the truncated constructs [2–41]V1aR and [2–44]V1aR had revealed a selective decrease in the affinity for AVP when the N-terminus was truncated from Pro41 to Asp44 (Table 1). Consequently, it was possible that the individual impact on agonist binding of residues Leu42, Gly43, and Asp44 was low but that when operating in concert they contributed to high affinity AVP binding. To address this further, the construct [L42A/G43A/D44A]V1aR was engineered, in which all three residues were substituted by alanyl. Pharmacological characterization of [L42A/G43A/D44A]V1aR revealed that simultaneous mutation of Leu42, Gly43, and Asp44 increased the Kd for AVP 8-fold (Fig. 2B) but did not affect the binding of any antagonist (Table 1). Consequently, AVP had a similar affinity for both [2–44]V1aR and for [L42A/G43A/D44A]V1aR (dissociation constant, Kd = 6.1 ± 1.5 nM and 7.9 ± 2.3 nM, respectively). These data suggest that, acting collectively, Leu42, Gly43, and Asp44 form a small patch within the N-terminus, which contributes to high affinity AVP binding but the individual contribution of residues within this patch is low.
Arg46 Is Critical for High Affinity Agonist Binding
Mutant constructs [V45A]V1aR and [N47A]V1aR possessed a pharmacological profile similar to wild-type V1aR (Fig. 2C and Table 1). In marked contrast, [R46A]V1aR exhibited a profound decrease in affinity for AVP with the Kd increasing 1,300-fold compared with wild-type receptor (Fig. 2C and Table 1). Substitution of Arg46 had no effect on the affinity of the receptor for the three different antagonists (Table 1). Consequently, the presence of Arg46 was critical for high affinity agonist binding but this residue did not have a role in binding antagonists of any class (cyclic or linear, peptide or nonpeptide). The decrease in AVP affinity which resulted from mutating the single residue in [R46A]V1aR was similar in magnitude to that observed by deleting forty-six residues in the N-terminally truncated construct [2–47]V1aR (Fig. 2, A and C; Table 1).
Role of the N-Terminal Domain of the V1aR in Second Messenger Generation
The ability of the truncated constructs to generate an intracellular signal in response to AVP was investigated. AVP-induced accumulation of inositol phosphates (InsPs) was measured and the dose-response curves for each receptor construct is presented in Fig. 3A. Both of the N-terminally truncated constructs [2–41]V1aR and [2–44]V1aR exhibited a slight increase (3-fold) in their EC50 values (concentration giving half-maximal response) relative to wild-type V1aR (Fig. 3A and Table 2). The maximum stimulation of InsPs production exhibited by each of these constructs was similar to wild-type values (Table 2). In contrast, the [2–47]V1aR construct failed to generate an intracellular signal when challenged by AVP (Fig. 3A and Table 2). These results suggested that a key locus for AVP-induced V1aR activation resided in the segment of the N-terminus between Val45 and Asn47. Whole cell binding assays using [3H]antagonist as tracer and HEK 293T cells expressing wild-type V1aR, [R46A]V1aR, or [2–47]V1aR revealed that all of these receptors exhibited the same cell surface expression (data not shown). Consequently, the lack of intracellular signal by [2–47]V1aR was not due to this construct failing to be trafficked efficiently to the plasma membrane.
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, B and C. With the exception of the [R46A]V1aR construct, the InsPs dose-response curves of single, and multiple, alanine-substituted constructs were essentially wild-type (Fig. 3, B and C, Table 2). In contrast, mutation of Arg46 had a marked effect on the InsPs dose-response curve. The curve was right-shifted with the EC50 value increased 65-fold compared with wild-type V1aR and the maximum response only 30% of wild-type (Table 2). It was noted that [G43A]V1aR also displayed a reduced maximum InsPs response to AVP (35% of wild-type), but this impaired signaling capability was not apparent with the triple mutant [L42A/G43A/D44A]V1aR (Table 2).
Specific Requirement for Arginyl at the Residue-46 Locus for High Affinity Agonist Binding and Second Messenger Generation
To evaluate the properties of the Arg46 residue that underlie its importance to V1aR function, we engineered the constructs [R46K]V1aR, [R46L]V1aR, and [R46E]V1aR. This series of constructs systematically probed the importance of different structural features of the arginyl side-chain, by either preserving the positive charge ([R46K]V1aR), mimicking the hydrophobic portion of the side-chain ([R46L]V1aR), or reversing the charge at this locus ([R46E]V1aR). In each case, the affinity of AVP was reduced 1,200- to 1,600-fold compared with wild-type V1aR (Fig. 4A and Table 3). In contrast, the binding affinities of the three different antagonists were unchanged compared with wild-type V1aR.
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). None of the substituted residues could support wild-type intracellular signaling. Maintaining the positive charge in the construct [R46K]V1aR resulted in a 90-fold increase in the EC50 compared with wildtype (Table 3). Introduction of a negative charge ([R46E]V1aR) was the most detrimental change at this locus and increased the EC50 for inositol phosphate accumulation 600-fold. In addition, the maximum response of [R46E]V1aR was only 36% of wild-type V1aR (Table 3). The second messenger generation characteristics of [R46L]V1aR were intermediate to those of [R46K]V1aR and [R46E]V1aR with an EC50 value 170-fold higher than wild-type. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The first objective of this study was to identify individual residues within the N-terminus that provided this agonist-specific interaction. Truncation of the N-terminus of the V1aR as far as Pro41 (construct [2–41]V1aR) resulted in a receptor that was essentially wild-type, indicating that residues from Ser2-Pro41 inclusive did not have a role in agonist/antagonist recognition, receptor folding or receptor activation. In addition, this indicated that the segment of the N-terminus required for agonist recognition was actually restricted to only six residues from Leu42-Asn47. Alanine-scanning mutagenesis over this region identified Arg46 as critical for high affinity binding of AVP. In contrast, Arg46 was not required for binding any of the three different classes of antagonists tested. The loss of agonist binding observed when this residue was deleted/substituted was not due to aberrant assembly of the receptor, or to local distortion of the mature protein, as the binding of both peptide and nonpeptide antagonists was largely unaffected. Moreover, this preservation of antagonist binding provided us with the means of accurately characterizing changes in agonist binding by using radioligand binding studies with [3H]antagonist as tracer. Binding of the cyclic antagonist was not affected by Arg46 substitution, despite this antagonist having an almost identical structure to AVP. The only differences between the two nonpeptides is that in the antagonist the Tyr2 is methylated and the Cys1 is replaced by ß-mercapto-ß,ß-cyclopentamethylenepropionate (23). Despite the high degree of structural homology between AVP and the cyclic antagonist, the molecular contacts between these two ligands and the receptor are different. AVP is an agonist, stabilizes an active conformation of the V1aR and requires Arg46. In contrast, the cyclic antagonist binds, does not induce receptor activation and is not dependent on Arg46. Systematic substitution of Arg46 was employed to evaluate the properties of the arginyl that are important for agonist binding and signaling. The constructs [R46K]V1aR and [R46L]V1aR provided the positive charge and hydrophobic side-chain elements of arginyl, respectively. However, neither of these individual elements of the arginyl side-chain in isolation were able to support wild-type agonist affinity or intracellular signaling. Reversing the nature of the charge at this locus was definitely detrimental. Consequently, [R46E]V1aR possessed reduced agonist affinity, exhibited a 64% decrease in maximum accumulation of InsPs in response to AVP and a 600-fold increase in EC50 (Table 3). Although the negative charge in [R46E]V1aR was very disruptive to receptor function, the positively charged lysyl 
-amino group in [R46K]V1aR could not mimic the wild-type arginyl’s guanidinium group (Table 3, Fig. 4). These data reveal that the structural requirements at this locus are not restricted to the charge alone. Both arginyl and lysyl have a charge on the end of a long chain but, despite this apparent similarity, they are very different. Lysyl is very flexible and mobile, whereas arginyl is only occasionally disordered. In addition, the guanidinium group of arginyl is unusual in having five hydrogen-bond donors held in a large planar array (26). These unique aspects of the arginyl side-chain at position-46 are a prerequisite for wild-type V1aR characteristics. The V1aR is a member of the rhodopsin/ß-adrenergic class of GPCRs. The recently published crystal structure of rhodopsin (27) revealed that the GPCR N-terminus was not random but formed a structured domain. Furthermore, the N-terminal strand of rhodopsin juxtaposed to the membrane, lay along the top of the helical bundle and covered the space between transmembrane helix (TM) I and II (27). The corresponding N-terminal strand of the V1aR contains Arg46. Molecular models of AVP docked to the V1aR (17, 20) show the C-terminus of AVP close to TMII of the receptor and, by analogy with the rhodopsin crystal structure, beneath the N-terminal strand of the V1aR containing Arg46. Therefore, it is plausible that Arg46 is required to make contact with the C-terminus of AVP.
An alignment of the N-terminal sequence of different receptors comprising the neurohypophysial peptide hormone receptor subfamily of GPCRs is shown in Fig. 5. For each subtype, the sequence of the receptor cloned from a range of different species is presented. All of these receptors are activated by AVP or its analogs. Within each receptor subtype, there are sequence motifs that are conserved across all of the species and that are characteristic of that particular subtype. However, there is no homology between the N-terminal sequence of different subtypes, even from the same species, which vary with respect to both length and identity. However, one notable exception is an arginyl located nine residues from the start of the first transmembrane domain that is absolutely conserved across all species and subtypes cloned to date (Fig. 5). In this study, we have established that this same arginyl (corresponding to Arg46 in the V1aR) is critical for the binding of AVP to the V1aR and intracellular signaling. Extrapolating from our findings, we would predict that this conserved extracellular arginyl residue has a fundamental role in agonist binding, and receptor activation, for all of the other members of the neurohypophysial peptide hormone receptor family. It is noteworthy in this regard that the N-terminal domain of both the vasotocin receptor (28) and of the OTR (29) has been implicated in ligand binding. Furthermore, a segment of the OTR N-terminus, which included the conserved arginyl, has been shown to provide agonist-specific binding epitopes (30). A key role in ligand binding of a single residue in the N-terminal domain has also been reported for the NK1 receptor. Substitution of Phe25 by Ala eliminated the binding of substance P, neurokinin A, and neurokinin B but did not affect the binding of the nonpeptide antagonist L-703,606 (31).
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2–44]V1aR and [L42A/G43A/D44A]V1aR constructs resulted in a decrease in AVP affinity of approximately 8-fold (Table 1). Alanine substitution revealed that the effect of each of these three residues individually was small but operating in concert they made a significant contribution to agonist binding (Table 1). A similar situation has been reported for the angiotensin AT1 receptor. Individual alanine substitution of His24, Tyr26, and Ile27 in the distal segment of the N-terminus of the AT1 receptor had only modest effects on angiotensin II binding. However, when mutated simultaneously in a triple alanine substitution, the affinity of angiotensin II decreased 3,000-fold (32). There is also a triplet of residues in a similar position in the N-terminus of the NK1 receptor (Asn23), Gln24, Phe25 that are required for high affinity substance P binding (31). Similarly, with the cholecystokinin-A (CCKA) receptor, a region of the N-terminus close to the first transmembrane helix was reported to be involved in CCK binding and residues Trp39 and Gln40 within this segment identified as forming part of the peptide binding site (33). For the large glycohormones such as choriogonadotropin and lutropin, the large size of the agonist requires a greater binding platform. In this case, there is such extensive interaction of the hormone with the N-terminus of its receptor that this domain expressed in isolation can bind choriogonadotropin (16).
We have shown previously that replacing the N-terminus of the V1aR with the corresponding sequence of the OTR generated a chimeric receptor (OTRN-V1aR) that exhibited high affinity AVP binding (22). The OTR sequence could not fully substitute for the V1aR sequence; however, as the OTRN-V1aR affinity was reduced (Kd = 7.5 ± 2.2 nM) compared with wild-type V1aR (Kd of 1.0 ± 0.1 nM). The molecular basis underlying this observation is explained by this study. The critical arginyl residue (
Arg46) was preserved in the OTRN-V1aR chimera, but the N-terminal OTR sequence of the chimera did not provide the proximal patch. The Leu42, Gly43, and Asp44 of the wild-type V1aR were replaced by Gly, Pro, and Pro in the OTR sequence. Consequently, the affinity of the OTRN-V1aR construct [Kd = 7.5 nM (22)] was the same as the affinity of the [2–44]V1aR and [L42A/G43A/D44A]V1aR constructs (Kd = 6.1 nM and 7.9 nM, respectively) reported in this study, which also lacked this patch (Table 1).
Both constructs lacking Arg46 (i.e. [R46A]V1aR and [2–47]V1aR) exhibited impaired intracellular signaling (Fig. 3, Table 2). When AVP-induced accumulation of InsPs was assayed, the [R46A]V1aR construct had an EC50 value 65-fold higher than wild-type V1aR and a maximum stimulation of InsPs production approximately one-third of wild type. The truncated receptor [2–47]V1aR also lacked Arg46 and exhibited disrupted InsPs production. However, a profound difference was observed between the [R46A]V1aR and the [2–47]V1aR constructs in that [2–47]V1aR failed to couple even when challenged by 10 µM AVP (compare Fig. 3, A and C; Table 2). Whole cell radioligand binding studies with [3H]antagonist showed that the cell surface expression of wild-type V1aR, [R46A]V1aR and [2–47]V1aR were identical (data not shown). Consequently, the lack of signaling by [2–47]V1aR was not due to defective trafficking of the construct to the plasma membrane. One difference between [R46A]V1aR and [2–47]V1aR is that the latter lacks the two glycosylation sites at Asn14 and Asn27. However, in a separate study, disruption of these glycosylation sites in wild-type V1aR by site-directed mutagenesis established that oligosaccharide modification did not affect either ligand binding or effector coupling (34). Given the affinity of [R46A]V1aR and [2–47]V1aR for AVP (Kd = 1.3 µM and 1.8 µM, respectively), basic receptor theory (35) dictates that 1 µM AVP would occupy 44% of the [R46A]V1aR and 36% of the [2–47]V1aR receptors. However, despite this similar level of receptor occupancy by 1 µM AVP, intracellular signaling by [R46A]V1aR was near maximal for this construct (Fig. 3C), whereas signaling by [2–47]V1aR was undetectable (Fig. 3A). These data established that, although the presence of Arg46 per se was pivotal to agonist binding in both [R46A]V1aR and [2–47]V1aR, the conformational context of the N-terminus surrounding Arg46 was also important for V1aR activation. Consequently, [R46A]V1aR could mediate an AVPinduced intracellular signal, albeit impaired, whereas [2–47]V1aR (which lacked an N-terminus) was incapable of signaling.
In conclusion, a series of V1aR constructs that possess truncations or alanine-substitutions within the N-terminus has been characterized. We have now identified that Arg46 has a critical role for high affinity agonist binding and receptor activation but not antagonist binding. Arg at this locus could not be replaced by Lys or by Leu and the negatively charged Glu was particularly detrimental. A cognate arginyl is absolutely conserved in all neurohypophysial peptide hormone receptors cloned to date, suggesting that this is a key residue for agonist:receptor interaction within this subfamily of GPCRs.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Truncated and Mutant Receptor Constructs
Truncations of the N-terminus of the V1aR were made using a PCR approach as described previously (22). Truncation oligonucleotides were 5'-G-GGG-GGG-CCC-GGA-TCC-GCC-ACC-ATG-CTG-GGG-GAC-GTA-CGC-AAT-GAG-G-3' and 5'-G-GGG-GGG-CCC-GGA-TCC-GCC-ACC-ATG-GTA-CGC-AAT-GAG-GAG-CTG-GCC-3' for the [2–41]V1aR and [2–44]V1aR truncations, respectively (Fig. 1). Each sense primer contained a BamHI restriction site (underlined), Kozak consensus sequence (shown in bold), and an ATG start site (shown in italics) followed by the V1aR sequence. The PCR products were subcloned into the rat V1aR coding sequence in the mammalian expression vector pcDNA3 (Invitrogen, Groningen, The Netherlands) utilizing unique BamHI and SdaI restriction sites. The mutant receptor constructs [L42A]V1aR, [G43A]V1aR and triple mutant [L42A/G43A/D44A]V1aR were also engineered using a PCR approach. Antisense oligonucleotides were 5'-C-CTC-ATT-GCG-TAC-GTC-CCC-TGC-GGG-GCT-GTC-ACC-3', 5'-C-CTC-ATT-GCG-TAC-GTC-CGC-TAA-GGG-GCT-GTC-ACC-3' and 5'-C-CTC-ATT-GCG-TAC-GGC-CGC-CGC-AGG-GCT-GTC-ACC-3' for the [L42A]V1aR, [G43A]V1aR and [L42A/G43A/D44A]V1aR mutations, respectively. These primers contained an unique Pfl23II restriction site (underlined) and the base changes (shown in bold) to incorporate the Ala substitutions at the desired positions within the receptor. The PCR products were subcloned into pcDNA3-V1aR vector utilizing unique HindIII and Pfl23II restriction sites.
The [R46A]V1aR was made by PCR using both sense and antisense oligonucleotides. The sense primer was 5'GGG-GAA-GGT-GAC-AGC-CCC-TTA-GGG-GAC-GTA-GCC-AAT-GAG-GAG-CTG-GCC-3'. This primer contained five base changes in the V1aR sequence (indicated in bold), which created an unique Eco81I restriction site (underlined) without altering the amino acid sequence and incorporated the Arg46
Ala mutation (shown in italics). A BamHI/SdaI digest of this PCR fragment was subcloned into the pcDNA3-V1aR vector. The constructs [D44A]V1aR, [V45A]V1aR, [N47A]V1aR, [R46L]V1aR, [R46K]V1aR and [R46E]V1aR were made using a PCR with pcDNA3-[R46A]V1aR as template. Mutant sense oligonucleotides were 5'-G-GGG-GCC-TTA-GGG-GCC-GTA-CGC-AAT-GAG-GAG-CTG-G-3', 5'-G-GGG-GCC-TTA-GGG-GAC-GCA-CGC-AAT-GAG-GAG-CTG-G-3', 5'-GGG-GCC-TTA-GGG-GAC-GTA-CGC-GCT-GAG-GAG-CTG-GCC-3', 5'-G-GGG-GCC-TTA-GGG-GAC-GTA-TTG-AAT-GAG-GAG-CTG-GCC-3', 5'-G-GGG-GCC-TTA-GGG-GAC-GTA-AAG-AAT-GAG-GAG-CTG-GCC-3' and 5'-G-GGG-GCC-TTA-GGG-GAC-GTA-GAG-AAT-GAG-GAG-CTG-GCC-3' for the [D44A]V1aR, [V45A]V1aR, [N47A]V1aR, [R46L]V1aR, [R46K]V1aR and [R46E]V1aR constructs, respectively. These primers contained the appropriate base changes (shown in bold) for the required substitution and the unique Eco81I restriction site (underlined). The PCR products were subcloned into pcDNA3-[R46A]V1aR vector utilizing unique Eco81I and SdaI restriction sites. All receptor constructs were confirmed by automated fluorescent sequencing (Alta Bioscience, University of Birmingham, Birmingham, UK).
Cell Culture and Transfection
HEK 293T cells were routinely cultured in DMEM supplemented with 10% (vol/vol) FCS, penicillin (100 IU/ml), streptomycin (100 µg/ml) in humidified 5% (vol/vol) CO2 in air at 37 C. Cells were seeded at a density of approximately 5 x 105 cells/100 mm dish and transfected after 48 h using a calcium phosphate precipitation protocol with 10 µg DNA/dish.
Radioligand Binding Assays
A washed cell membrane preparation of HEK 293T cells, transfected with the appropriate receptor construct, was prepared as previously described (36) and the protein concentration determined using the BCA protein assay kit (Pierce Chemical Co., Tattenhall, Cheshire, UK) with BSA as standard. Radioligand binding assays were performed as previously described (37) using either the natural agonist [Phe3-3,4,5-3H]AVP, (64.2 Ci/mmol; NEN Life Science Products, Stevenage, Herts., UK) or the V1aR-selective peptide antagonist [Phe3-3,4,5-3H] d(CH2)5Tyr(Me)2AVP (99 Ci/mmol; NEN Life Science Products) (23) as tracer ligand. Binding data were analyzed by nonlinear regression to fit theoretical Langmuir binding isotherms to the experimental data using the Fig. P program (Biosoft, Cambridge, UK). Individual IC50 values obtained for competing ligands were corrected for radioligand occupancy as described (38) using the radioligand affinity (Kd) experimentally determined for each construct. Cell surface expression was determined by whole cell binding assays as described in (39). Briefly, HEK 293T cells were seeded at a density of 1 x 105 cells/well in poly D-lysine-coated 12-well plates and transfected after 24 h using Transfast (Promega Corp., Southampton, UK). After 36 h, cells were washed twice with PBS, after which each well received 0.5 ml of binding buffer containing 2% (wt/vol) BSA, [3H]d(CH2)5Tyr(Me)2AVP in the presence (nonspecific) and absence (total) of 1 µM d(CH2)5Tyr(Me)2AVP. Plates were incubated for 90 min at 30 C before removal of the medium by aspiration. After two rinses with ice-cold PBS, 0.5 ml of 0.1 M NaOH was added to each well to extract radioactivity. After 15 min incubation at 37 C, the medium from the plates was transferred to scintillation vials containing 10 ml HiSafe3 scintillation cocktail.
AVP-Induced Inositol Phosphate Production
HEK 293T cells were seeded at a density of 2.5 x 105 cells/well in poly D-lysine-coated 12-well plates and transfected after 24 h using Transfast (Promega Corp.). The assay for AVP-induced accumulation of InsPs was based on that described previously (30, 40). Essentially, 16 h post transfection, medium was replaced with inositol-free DMEM containing 1% (vol/vol) FCS and 2 µCi/ml myo-[2-3H]inositol (22.0 Ci/mmol; NEN Life Science Products) for 24 h. Cells were washed twice with PBS, then incubated in inositol-free medium containing 10 mM LiCl for 30 min, after which AVP was added at the concentrations indicated for a further 30 min. Incubations were terminated by adding 0.5 ml of 5% (vol/vol) perchloric acid containing 1 mM EDTA and 1 mg/ml phytic acid hydrolysate. Samples were neutralized with 1.2 M KOH, 10 mM EDTA, 50 mM HEPES on ice for 1 h, insoluble material sedimented at 12,000 x g for 5 min and supernatants loaded onto Bio-Rad Laboratories, Inc. (Hemel Hempstead, Herts., UK) AG1-X8 columns (formate form) in 10 ml of water. After the elution of inositol and glycerophosphoinositol (10 ml of 25 mM NH4COOH containing 0.1 M HCOOH), a mixed inositol fraction containing mono-, bis-, and trisphosphates (InsP-InsP3) was eluted with 10 ml of 850 mM NH4COOH containing 0.1 M HCOOH, mixed with UltimaFlo AF scintillation cocktail (Packard, Pangbourne, Berks., UK) and radioactivity quantified by liquid scintillation spectroscopy. EC50 values were determined by nonlinear regression after fitting of logistic sigmoidal curves to the experimental data.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: EC50, Concentration giving half-maximal response; GPCR, G protein-coupled receptor; InsPs, inositol phosphates; Kd, dissociation constant; OTR, oxytocin receptor; V1aR, vasopressin V1a receptor; V1bR, V1b receptor; V2R, V2 receptor; VPR, vasopressin receptor.
Received for publication August 16, 2001. Accepted for publication November 27, 2001.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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S. R. Hawtin, J. Simms, M. Conner, Z. Lawson, R. A. Parslow, J. Trim, A. Sheppard, and M. Wheatley Charged Extracellular Residues, Conserved throughout a G-protein-coupled Receptor Family, Are Required for Ligand Binding, Receptor Activation, and Cell-surface Expression J. Biol. Chem., December 15, 2006; 281(50): 38478 - 38488. [Abstract] [Full Text] [PDF]
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S. R. Hawtin Pharmacological Chaperone Activity of SR49059 to Functionally Recover Misfolded Mutations of the Vasopressin V1a Receptor J. Biol. Chem., May 26, 2006; 281(21): 14604 - 14614. [Abstract] [Full Text] [PDF]
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 S. R. Hawtin, V. J. Wesley, J. Simms, C. C. H. Argent, K. Latif, and M. Wheatley The N-Terminal Juxtamembrane Segment of the V1a Vasopressin Receptor Provides Two Independent Epitopes Required for High-Affinity Agonist Binding and Signaling Mol. Endocrinol., November 1, 2005; 19(11): 2871 - 2881. [Abstract] [Full Text] [PDF]
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 S. R. Hawtin Charged Residues of the Conserved DRY Triplet of the Vasopressin V1a Receptor Provide Molecular Determinants for Cell Surface Delivery and Internalization Mol. Pharmacol., October 1, 2005; 68(4): 1172 - 1182. [Abstract] [Full Text] [PDF]
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 A Levoye, B Mouillac, G Riviere, D Vieau, M Salzet, and C Breton Cloning, expression and pharmacological characterization of a vasopressin-related receptor in an annelid, the leech Theromyzon tessulatum J. Endocrinol., January 1, 2005; 184(1): 277 - 289. [Abstract] [Full Text] [PDF]
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S. Acharjee, J.-L. Do-Rego, D. Y. Oh, R. S. Ahn, H. Choe, H. Vaudry, K. Kim, J. Y. Seong, and H. B. Kwon Identification of Amino Acid Residues That Direct Differential Ligand Selectivity of Mammalian and Nonmammalian V1a Type Receptors for Arginine Vasopressin and Vasotocin: INSIGHTS INTO MOLECULAR COEVOLUTION OF V1a TYPE RECEPTORS AND THEIR LIGANDS J. Biol. Chem., December 24, 2004; 279(52): 54445 - 54453. [Abstract] [Full Text] [PDF]
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S. Derick, A. Pena, T. Durroux, J. Wagnon, C. Serradeil-Le Gal, M. Hibert, D. Rognan, and G. Guillon Key Amino Acids Located within the Transmembrane Domains 5 and 7 Account for the Pharmacological Specificity of the Human V1b Vasopressin Receptor Mol. Endocrinol., November 1, 2004; 18(11): 2777 - 2789. [Abstract] [Full Text] [PDF]
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C. Tahtaoui, M.-N. Balestre, P. Klotz, D. Rognan, C. Barberis, B. Mouillac, and M. Hibert Identification of the Binding Sites of the SR49059 Nonpeptide Antagonist into the V1a Vasopressin Receptor Using Sulfydryl-reactive Ligands and Cysteine Mutants as Chemical Sensors J. Biol. Chem., October 10, 2003; 278(41): 40010 - 40019. [Abstract] [Full Text] [PDF]
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); [
); [
); or [
); B, wild-type V1aR, (
); G43A, (
); D44A, (
); or L42A/G43A/D44A, (
); and C, wild-type V1aR, (
); R46A, (
); or N47A, (
). Data are the mean ± SEM of three separate experiments each performed in triplicate. Values are expressed as percent specific binding where nonspecific binding was defined by d(CH2)5Tyr(Me)2AVP (1 µM). A theoretical Langmuir binding isotherm has been fitted to the experimental data as described in Materials and Methods.
