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Am J Physiol Gastrointest Liver Physiol 291: G728-G734, 2006; doi:10.1152/ajpgi.00138.2006
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HORMONES AND SIGNALING

Molecular cloning and functional expression of a VIP-specific receptor

Huiping Zhou, Jiean Huang, and Karnam S. Murthy

Departments of Physiology and Medicine, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Virginia

Submitted 27 March 2006 ; accepted in final form 8 May 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Three receptors for VIP and pituitary adenylate cyclase-activating peptide (PACAP) have been cloned and characterized: PAC1, with high affinity for PACAP, and VPAC1 and VPAC2 with equally high affinity for VIP and PACAP. The existence of a VIP-specific receptor (VIPs) in guinea pig (GP) teniae coli smooth muscle was previously surmised on the basis of functional studies, and its existence was confirmed by cloning of a partial NH2-terminal sequence. Here we report the cloning of the full-length cDNAs of two receptors, a VPAC2 receptor from GP gastric smooth muscle and VIPs from GP teniae coli smooth muscle. The cDNA sequence of the VIPs encodes a 437-amino acid protein (Mr 49,560) that possesses 87% similarity to VPAC2 receptors in rat and mouse and differs from the VPAC2 receptor in GP gastric smooth muscle by only two amino-acid residues, F40F41 in lieu of L40L41. In COS-1 cells transfected with the GP teniae coli smooth muscle receptor, only VIP bound with high affinity (IC50 1.4 nM) and stimulated cAMP formation with high potency (EC50 1 nM). In contrast, in COS-1 cells transfected with the GP gastric smooth muscle receptor, both VIP and PACAP bound with equally high affinity (IC50 2.3 nM) and stimulated cAMP with equally high potency (EC50 1.5 nM). We conclude that the receptor cloned from GP teniae coli smooth muscle is a VIPs distinct from VPAC1 and VPAC2 receptors. The ligand specificity in this species is determined by a pair of adjacent phenylalanine residues (L40L41) in the NH2-terminal ligand-binding domain.

VIP and pituitary adenylate cyclase-activating peptide (PACAP) are homologous, regulatory neuropeptides present in neurons of the central and peripheral nervous systems. Both peptides are abundantly expressed in neurons of the enteric nervous system where they regulate intestinal secretion and, together with nitric oxide (NO), descending inhibitory reflexes and peristaltic activity (1, 2, 4–6, 13, 18, 21). The biological effects of VIP and PACAP are mediated by cognate seven-transmembrane receptors coupled via Gs to activation of adenylyl cyclase; in smooth muscle of the gut, both peptides interact additionally with single-transmembrane natriuretic peptide receptor, NPR-C, coupled via Gi1 and Gi2 to activate NO synthase (14, 15, 27). Three PACAP/VIP receptors have been cloned so far from various tissues and cell lines in rat and human: PAC1, a receptor with high affinity for PACAP and very low affinity (1,000-fold less) for VIP, and VPAC1 and VPAC2 with equally high affinity for VIP and PACAP (7, 21, 23, 25). Five splice variants of PAC1 have been identified in rat and human (19, 20, 22). They exhibit variable tissue distribution and are coupled with different affinities to both Gs and Gq, leading to differential activation of adenylyl cyclase and phospholipase C-beta (19, 22).

A VIP-specific receptor (VIPs) with high affinity for VIP but not for PACAP has not been cloned yet. Its existence, however, has been surmised from functional and molecular studies in smooth muscle of guinea pig (GP) teniae coli (8, 16, 24). We have previously cloned the NH2-terminal partial sequence of a VIPs from GP teniae coli smooth muscle cells (24). When expressed in COS-1 cells, chimeric receptors consisting of the rat VPAC2 receptor with its NH2-terminal extracellular domain replaced by the partial sequence cloned from GP teniae muscle cells exhibited specificity for VIP. Chimeric receptors in which the NH2-terminal domain of rat VPAC2 was replaced by the corresponding sequence cloned from GP gastric muscle cells exhibited equal affinity for VIP and PACAP. Site-directed mutagenesis suggested that the specificity of the VIP-receptor derived from teniae coli might reside in a pair of extracellular phenylalanine residues (24).

We have now cloned the full-length cDNA sequences of these receptors from GP gastric and teniae coli smooth muscle cells and expressed them separately in COS-1 cells. The functional characteristics of both receptors were determined by ligand binding and cAMP formation. The receptor cloned from GP gastric smooth muscle differed in sequence but was functionally identical to VPAC2 receptors cloned from other species and exhibited equally high affinity for VIP and PACAP. The receptor cloned from GP teniae coli exhibited high affinity for VIP but not for PACAP. The amino acid sequence of the receptor cloned from GP teniae coli differed from the sequence of the receptor cloned from GP gastric smooth muscle by two adjacent phenylalanine residues (F40F41) in the ligand-binding extracellular domain.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
cDNA library construction. Two cDNA libraries were constructed from the cultured GP teniae coli and gastric smooth muscle cells using the ZAP Express cDNA synthesis kit. Briefly, mRNAs were isolated from teniae coli and gastric smooth muscle cells using the Poly(A)Pure mRNA isolation kit. The first-strand cDNA was synthesized using oligo(dT) primer and was used as template to synthesize the second-strand cDNA. After addition of adapters with unique restriction enzyme sites at both 5' and 3' ends of the second-strand cDNA, the cDNA was ligated into ZAP Express vector and packaged into lambda phage in vitro using a ZAP Express cDNA Gigapack III gold cloning kit.

Isolation of cDNA clones. The partial cDNA sequence of the receptor isolated from GP teniae coli by RT-PCR was 32P labeled using a Prime-It II random primer labeling kit and was used as a probe to screen the cDNA library by plaque hybridization as described previously (26). A total of 6 x 105 phages were plated, transferred to nylon membranes, and screened by hybridization at 42°C for 16 h in 5x sodium chloride-sodium phosphate-EDTA (SSPE) (0.75 M NaCl, 50 mM sodium phosphate, 6 mM EDTA, pH 7.4), 50% formamide, 5x Denhardt's solution, 0.1% SDS, 100 ng/ml of heat-denatured salmon sperm DNA, and the radiolabled probe (2 x 106 cpm/ml). Filters were washed for 30 min at 65°C three times with 2x SSPE containing 0.1% SDS. Several positive clones were plaque purified and verified by DNA sequencing.

5-rapid amplification of cDNA ends. A Marathon cDNA amplification kit was used to amplify the NH2-terminal coding region, and 1 µg of poly-A RNA was used for cDNA amplification. After the cDNAs were synthesized, a gene-specific primer (antisense, 5'-ATC TGG GAA TGT CTC TGA CCA TCC-3') was used together with primer AP1 (Clontech) to perform the 5-rapid amplification of cDNA ends (RACE) reaction using touchdown PCR. The conditions for touchdown PCR were 30 s at 94°C, 5 s at 94°C, and 4 min at 72°C for 5 cycles; 5 s at 94°C and 4 min at 70°C for 5 cycles; then 5 s at 94°C and 4 min at 68°C for 25 cycles. 5-RACE products were cloned into pCRII-TOPO vector using the TOPO TA Cloning kit and were verified by DNA sequencing.

PCR cloning of the full-length cDNA. The lambda DNA was isolated from GP teniae coli and gastric smooth muscle cell cDNA libraries using the Lambda mini kit and was used as templates for PCR amplification of the full coding region of the receptors. On the basis of the DNA sequence information from several positive clones isolated from the cross-hybridization screening of the cDNA libraries and the 5-RACE described above, two primers were designed (sense primer, 5'-ATG AGG GCG TCG GTG GTG CTG-3'; antisense primer, 5'-CTA AAT GAC TGA GGT CTC-3') and used to amplify the full coding regions of the receptors. The PCR products were cloned into pCRII-TOPO vector using the TOPO TA Cloning kit and were verified by DNA sequencing.

Expression of the cloned VIP receptors in COS-1 cells. The full-length cDNAs of the cloned receptors from GP teniae coli and gastric smooth muscle cells were subcloned into the mammalian expression vector pcDNA3.1 at BamHI and EcoRI sites. COS-1 cells were grown at 37°C in a humidified atmosphere of 95% air and 5% CO2 in DMEM with 10% FBS, penicillin (50 U/ml), streptomycin (50 µg/ml), and gentamicin (100 µg/ml). Two micrograms of pcDNA3.1 containing the full-length cDNA insert were transfected into COS-1 cells using Effectene transfection reagent. Transfected cells were isolated in a medium containing 500 µg/ml geneticin (G418). G418-resistant cells were subcultured, and confluent monolayers were screened for expression of receptors by radioligand binding using [125I]VIP. Receptor expression was verified by Western blot using antibodies raised against the COOH terminal of VPAC2 receptors. Selection pressure for clonal cell lines was maintained by the addition of 100 µg/ml G418 to the culture medium. For all studies, transfected cells in monolayers at ~90% confluence were utilized.

[125I]VIP binding assay. The binding assay was performed as described previously (14, 24). Confluent cultures of COS-1 cells stably transfected with the cloned receptors were detached with 0.25% trypsin and 1 mM EDTA. Cells were immediately centrifuged at 500 g for 5 min at 4°C. The cell pellet was washed with PBS and homogenized in smooth muscle buffer (SMB) containing 1 µM phosphoramidon and 10 µM amastatin. The homogenate was centrifuged at 30,000 g for 30 min at 4°C, and the membrane pellet was resuspended in SMB buffer containing 1% BSA, 1 µM phosphoramidon, and 10 µM amastatin. Homogenate membranes (50 µg) from cells expressing the receptor were incubated with 50 pM 125I-labeled VIP at 25°C for 30 min in the presence or absence of various concentrations of unlabeled VIP or PACAP-38. The reaction was terminated by addition of 3 ml of ice-cold PBS. The solution was filtered under vacuum through a Whatman GF/C glass filter, and the filter was washed three times with 5 ml of PBS containing 0.1% BSA. The radioactivity retained on the filter was counted, and the specific binding was calculated as the difference between total binding and nonspecific binding (28 ± 5%) measured in the presence of 10 µM VIP.

cAMP assay. cAMP was measured by radioimmunoassay as described previously (14, 24). Transfected COS-1 cells (106 cells/0.5 ml) were detached and incubated for 60 s in the presence of 100 µM 3-isobutyl-L-methylxanthine with or without the indicated concentration of VIP or PACAP-38. The reaction was terminated with 100 µl of 10% ice-cold trichloroacetic acid, and cAMP was extracted by freeze thawing. The acid was removed by ether extraction, and cAMP was measured in duplicate by radioimmunoassay using 100-µl aliquots of reconstituted samples. The results were expressed in picomoles per milligram of protein.

Materials. pcDNA3.1 vector, Platinum Taq DNA polymerase, and TOPO TA Cloning kit were from Invitrogen (Carlsbad, CA); ZAP Express cDNA synthesis kit, ZAP Express cDNA Gigapack III gold cloning kit, and Primer-It II random primer labeling kit were from Stratagene (La Jolla, CA); Marathon cDNA amplification kit was from Clontech (Palo Alto, CA); Effectene transfection reagent, Lambda mini kit, and plasmid DNA purification kit were from QIAGEN (Valencia, CA); Poly(A)Pure mRNA isolation kit was from Ambion (Austin, TX); T4 DNA ligase, BamHI, and EcoRI were from New England Biolabs (Beverly, MA); VIP and PACAP were from Bachem (Torrance, CA); and 125I-labeled VIP, 125I-labeled cAMP, and [{gamma}-32P]dCTP were from New England Nuclear (Boston, MA); all other chemicals were from Sigma, oligonucleotides were synthesized by Integrated DNA Technologies (Coralville, IA), and DNA sequencing was done by the Virginia Commonwealth University Nucleic Acid Core Facility.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Cloning of distinct receptor cDNAs from GP teniae coli and gastric smooth muscle cells. To isolate the full-length cDNA of teniae coli receptor, a cDNA library was constructed with the mRNA from cultured GP teniae coli smooth muscle cells using ZAP Express vector. The library was screened under high-stringency conditions using the NH2-terminal partial cDNA sequence of the GP teniae coli VIP receptor as probe. Several positive clones were isolated and purified by subsequent rounds of screening, and the insert was identified by DNA sequencing. None of the positive clones contained the full coding sequence. The longest clone contained 1,260 base pairs, which included the COOH-terminal stop codon. However, the first 17-amino acid sequence on the NH2 terminal was missing. To isolate the NH2-terminal sequence, 5-RACE was performed. Two gene-specific primers based on the cloned partial cDNA sequence were designed and used to amplify the 5'-region sequence. The 5-RACE products were verified by DNA sequencing. On the basis of the results of the 5-RACE and cDNA library screening, the full-length cDNA was cloned by end-to-end PCR using 5'- and 3'-end gene-specific primers and was cloned into pCRII-TOPO vector. The cloned full-length cDNA was fully sequenced. The results showed that the cloned cDNA sequence contained an open reading frame that codes for a protein of 437 amino acids with a calculated Mr of 49,560 (Fig. 1). The amino acid sequence of the cloned receptor from GP teniae coli had 87% similarity with rat and mouse VPAC2 receptors (Fig. 2).


Figure 1
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  		Fig. 1. Nucleotide and deduced amino acid sequences of the VIP receptor cloned from guinea pig (GP) teniae coli. The nucleotide sequence has been submitted to the Gene Bank (accession number AY584606). Numbering of the nucleotide sequence begins with the A of the initiation codon (ATG). The deduced amino acid sequence is shown beneath the nucleotide sequence. The start and stop codons are underlined.

 

Figure 2
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  		Fig. 2. Amino-acid sequence alignment of GP teniae coli (GPT) and gastric (GPG) receptors and rat VIP adenylate cyclase-activating type 2 receptor (RAT). The amino acid sequence of GPT has been submitted to Gene Bank (accession number AY584607). The only difference between the GPT and GPG receptors is the presence of adjacent phenylalanine residues (F40F41) in lieu of leucine residues (L40L41). There is 87% similarity between GP and rat receptors.

 
A similar approach was used to clone the receptor cDNA from cultured gastric smooth muscle cells. The NH2-terminal partial cDNA sequence of the GP gastric VIP receptor was used as a probe to screen the GP gastric cDNA library under high-stringency conditions. Several positive clones were isolated and purified by subsequent rounds of screening, and the insert was identified by DNA sequencing. One positive clone contained partial cDNA sequence including the NH2-terminal start code, and several positive clones contained COOH-terminal partial sequence including the stop code. On the basis of the NH2-terminal and COOH-terminal sequences, we designed two primers to amplify the full-length cDNA sequence using the GP gastric cDNA library as template by PCR. The PCR product was cloned into pCRII-TOPO vector and fully sequenced. The results showed that the cloned gastric VPAC2 receptor cDNA sequence differs from the cloned teniae coli VIP receptor by only two amino acids in the NH2-terminal ligand-binding domain (F40F41 to L40L41) (Fig. 2).

Ligand-binding specificity of the receptors cloned from GP teniae coli and gastric smooth muscle. The cloned GP teniae coli and gastric smooth muscle receptors were separately expressed in COS-1 cells by stable transfection (27). Receptor expression was verified by Western blot using antibodies raised against the COOH terminal of VPAC2 receptors. The antibody recognized both cloned GP teniae and gastric smooth muscle recepors (data not shown). The specificities of 125I-labeled VIP binding to the cloned receptors were characterized separately using plasma membrane fractions. 125I-labeled VIP binding to the cloned GP teniae receptor was specifically inhibited by VIP in a concentration-dependent fashion with an IC50 of 1.4 nM, whereas inhibition by PACAP-38 was minimal (<105) (Fig. 3A). In contrast, 125I-labeled VIP binding to the cloned gastric smooth muscle receptor was inhibited with identical affinity by VIP and PACAP-38 (IC50 2.3 nM) (Fig. 3B).


Figure 3
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  		Fig. 3. [125I]VIP binding to membranes from COS-1 cells stably transfected with the cloned GPT or GPG receptors. Cell membranes derived from transfected COS-1 cells were incubated with [125I]VIP (50 pM) either alone or with increasing concentrations of VIP or pituitary adenylate cyclase-activating peptide (PACAP)-38. Nonspecific binding (28 ± 5%) was determined in the presence of 10 µM VIP. A: COS-1 cells transfected with receptor cDNA cloned from GPT smooth muscle cells. B: COS-1 cells transfected with receptor cDNA cloned from GPG smooth muscle cells. Data are presented as percentages of control specific binding (total binding in the presence of [125I]VIP alone minus binding in the presence of 10 µM VIP). Values are means ± SE of 5 experiments.

 
Stimulation of cAMP formation. Activation of adenylyl cyclase by VIP and PACAP-38 was determined as a functional correlate of receptor binding. In COS-1 cells stably transfected with the cloned GP teniae coli receptor, VIP stimulated cAMP formation in a concentration-dependent fashion with an EC50 of 1 nM, whereas PACAP-38 was less potent and had an efficacy of ~20% relative to VIP (Fig. 4A). In contrast, in COS-1 cells transfected with the cloned GP gastric smooth muscle receptor, both VIP and PACAP-38 stimulated cAMP formation in a concentration-dependent fashion with equally high potency (EC50 1.5 nM) (Fig. 4B).


Figure 4
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  		Fig. 4. Cyclic AMP formation in response to VIP and PACAP in COS-1 cells transfected with cDNA cloned from GPT or GPG smooth muscle cells. Transfected COS-1 cells were incubated with different concentrations of VIP or PACAP-38. cAMP was measured by radioimmunoassay, and the results are expressed as pmol/mg protein above basal levels (21 ± 5 pmol/mg protein). A: COS-1 cells transfected with cDNA cloned from GPT smooth muscle cells. B: COS-1 cells transfected with cDNA cloned from GPG smooth muscle cells. Values are means ± SE of 3–5 experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we report the molecular cloning of the full-length cDNA of two receptors from GP smooth muscle, a VIPs from GP teniae coli smooth muscle cells and a VPAC2 receptor from GP gastric smooth muscle cells. We have labeled the VIP-specific receptor as VIPs, to emphasize its specificity for VIP and to distinguish it from VPAC1 or VPAC2 receptors. The cDNA sequence of the VIPs confirmed from several clones encodes a 437-amino acid protein (Mr 49,560) with 87% similarity to the sequences of rat and mouse VPAC2 receptors and differed from the VPAC2 receptor in GP gastric smooth muscle cells by only two amino-acid residues, Phe40 and Phe41, in the NH2-terminal extracellular ligand-binding domain. In COS-1 cells stably expressing the VIPs, VIP, but not PACAP, bound with high affinity to the receptor and stimulated cAMP formation with high potency. In contrast, in COS-1 cells stably expressing the cloned GP gastric smooth muscle receptor, VIP and PACAP bound with equally high affinity and stimulated cAMP with equally high potency. The receptor cloned from GP gastric smooth muscle possesses the same characteristics as the VPAC2 receptor expressed in smooth muscle from other species (7, 23).

Similar results were previously obtained using chimeric receptors comprising the COOH-terminal sequence of the rat VPAC2 receptor coupled to the NH2-terminal ligand binding domain of the receptor from GP teniae coli or gastric smooth muscle cells (24). In COS-1 cells expressing the chimeric rat/GP teniae coli receptor, VIP but not PACAP bound the receptor with high affinity and stimulated cAMP with high potency. In contrast, in COS-1 cells expressing the chimeric rat/GP gastric receptor, both VIP and PACAP bound the receptor with equally high affinity and stimulated cAMP with equally high potency. The only difference between the chimeric receptors was the presence of a pair of adjacent phenylalanine residues (F40F41) in the NH2-terminal sequence cloned from GP teniae in lieu of leucine residues (L40L41) in the sequence cloned from GP gastric smooth muscle; adjacent leucine residues in these positions commonly occur in VPAC2 and VPAC1 receptors cloned from other tissues or species (11, 12, 23). It thus seems that the presence of a pair of phenylalanine residues in the NH2-terminal domain of the GP teniae coli receptor determines specific recognition of, and activation by, VIP. However, mutation of L40L41 to F40F41 in rat VPAC2 receptors did not yield specificity for VIP, implying that other residues in the NH2-terminal domain of rat VPAC2 receptors that are not shared by the GP gastric or teniae coli smooth muscle receptors maintain the ability of the rat receptor to recognize both VIP and PACAP.

The existence of a distinct VIPs in teniae coli was surmised from earlier functional studies (8, 16). VIP and PACAP activate distinct receptors that mediate relaxation in muscle strips and dispersed smooth muscle cells from teniae coli. VIP stimulated cAMP formation, whereas PACAP, despite its name, did not. VIP-induced relaxation was selectively inhibited by the cAMP-dependent protein kinase inhibitor H-89 and by the VIP antagonist VIP10–28, whereas PACAP-induced relaxation was selectively inhibited by the PACAP antagonist PACAP6–28 and by the K+-channel blocker apamin. Receptor desensitization after 30-min exposure to VIP or PACAP selectively blocked the response to the corresponding peptide. Similar results were obtained following endogenous release of VIP and PACAP by nerve stimulation of teniae coli muscle strips; the resultant relaxation was inhibited in equal measure by PACAP6–38, PACAP receptor desensitization, and apamin, and each of these effects was additive to those of VIP10–28 or VIP receptor desensitization, providing further evidence that VIP and PACAP interact with distinct receptors (8). The PACAP-specific receptor in teniae coli has not been cloned but could be a variant of PAC1 receptors. A TM4 variant of PAC1 receptors expressed in beta-islets cells mediates Ca2+ influx, which could, in turn, activate apamin-sensitive, small conductance K+ channels (3). The existence of apamin-sensitive PACAP receptors has been reported in various smooth muscle tissues in different species (8, 9).

In summary, we have cloned a VIPs from GP teniae coli smooth muscle that differs from the corresponding VPAC2 receptor in GP gastric smooth muscle by two adjacent amino acid residues (F40F41) in the extracellular ligand-binding domain. The two residues determine VIP specificity in this species.


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This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-28300.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. S. Murthy, Depts. of Physiology and Medicine, Medical College of Virginia Campus, VA Commonwealth Univ., Richmond, VA 23298 (e-mail: skarnam{at}hsc.vcu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
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