©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Critical Contributions of Amino-terminal Extracellular Domains in Agonist Binding and Activation of Secretin and Vasoactive Intestinal Polypeptide Receptors
STUDIES OF CHIMERIC RECEPTORS (*)

Martin H. Holtmann , Elizabeth M. Hadac , Laurence J. Miller (§)

From the (1)Center for Basic Research in Digestive Diseases, Mayo Clinic and Foundation, Rochester, Minnesota 55905

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Secretin and vasoactive intestinal polypeptide (VIP) receptors are closely related G protein-coupled receptors in a recently described family possessing a large amino-terminal ectodomain. We postulated that this domain might be critical for agonist recognition and therefore constructed a series of six chimeric receptors, exchanging the amino terminus, the first extracellular loop, or both in secretin and VIP receptors. Constructs were expressed in COS cells and characterized by cAMP generation and binding of both secretin and VIP radioligands. Wild type receptors demonstrated high affinity binding of respective ligands (IC values (in nM): at the secretin receptor: 2.2 for secretin, >1000 for VIP; at the VIP receptor: 2.2 for VIP, >1000 for secretin) and appropriately sensitive and selective biological responses (EC values (in nM): at the secretin receptor: 1.5 for secretin, 127 for VIP; at the VIP receptor: 1.0 for VIP, 273 for secretin). Replacement of the secretin receptor amino terminus with that of the VIP receptor resulted in biological responsiveness typical of the VIP receptor (EC = 120 nM for secretin, 1.7 nM for VIP). The converse was not true, with this domain of the secretin receptor not able to provide the same response when incorporated into the VIP receptor (EC = 50 nM for VIP, 30 nM for secretin). The addition of both the first loop and the amino terminus of the secretin receptor was effective in yielding a secretin receptor-like response (EC = 2.0 nM for secretin, 47 nM for VIP). All chimeric constructs expressing selectivity for secretin-stimulated activity bound this hormone with high affinity (IC = 0.2-2.2 nM); however, there was divergence between VIP binding and biological activity. Thus, the amino terminus of secretin and VIP receptors plays a key role in agonist recognition and responsiveness, with the first loop playing a critical complementary role for the secretin receptor.


INTRODUCTION

Secretin and vasoactive intestinal polypeptide (VIP)()receptors belong to a recently described family of G protein-coupled receptors (1, 2, 3) that are only 12% homologous with other members of this superfamily and lack the signature sequences that are conserved in the -adrenergic receptor family. Notable features of this group include a long extracellular amino terminus and a series of 8 conserved cysteine residues in the predicted ectodomain. Of interest, ligands for these receptors tend to be moderately large peptides having diffuse pharmacophoric domains(4) . A central hypothesis for the current work is that these large and complex domains interact with each other.

The ligand-binding domains of several G protein-coupled receptors have been well explored(5, 6) . Investigations have largely focused on members of the -adrenergic receptor family, which bind very small molecules in the outer third of the plasma membrane between transmembrane helices(5) . Receptors in this family that bind small peptide ligands have also been examined recently, suggesting a theme that proximal loop regions are important binding determinants(7) . Binding themes have also been established for the metabotropic glutamate receptors, the glycoprotein hormone receptors, and the thrombin receptor(6) .

To date, however, few details exist regarding molecular mechanisms of ligand binding and receptor activation in the calcitonin-parathyroid hormone receptor family(8, 9) . In this work, we focus on the receptors for secretin and VIP that are 44% identical in sequence (10, 11, 12) and signal similarly(13) . In spite of their structural and functional similarities, however, they display distinct specificity for agonist ligands. This makes them ideal candidates for analysis using chimeric receptor proteins.

Using this approach, we have demonstrated that 1) the extracellular amino terminus of the VIP receptor is the critical domain responsible for the selectivity of activation by VIP; 2) the same domain of the secretin receptor is also important for the selectivity of high affinity binding and activation by secretin but is not sufficient by itself to provide this function; 3) the first extracellular loop of the secretin receptor is also a key domain for selective agonist binding and activation; and 4) in this series of constructs high affinity secretin binding correlated well with secretin-like biological activity, whereas VIP binding could be dissociated from its biological responses.


MATERIALS AND METHODS

We cloned the secretin receptor cDNA from a rat pancreatic cDNA library (11) and acquired the rat pancreatic VIP receptor cDNA from Professor Nagata (Osaka Bioscience Institute, Osaka, Japan). Synthetic rat secretin and VIP were from Peninsula Laboratories. [Tyr,pNO-Phe]rat secretin 27 was synthesized as we described(11) . All other reagents were analytical grade.

Receptor Constructs

The chimeric receptors utilized in this study include portions of wild type rat secretin and VIP receptors (). Chimeras were constructed by excising sequences of the wild type receptor cDNAs and replacing them with the corresponding sequences of the other receptor. Naturally occurring restriction sites were used when possible; otherwise PCR mutagenesis was performed.

VeS

This chimera was generated using a unique restriction site for BstXI present in analogous positions in VIP receptor (SLASLLVA) and secretin receptor (SLAMLLVA) cDNAs. The HindIII-BstXI fragment of the VIP receptor cDNA was ligated into the secretin receptor cDNA.

SeV

The VIP receptor sequence between codons 117 and 429 was amplified by PCR introducing a new restriction site for BsrGI at the 5` end and a stop codon and HindIII site at the 3` end. The PCR product was then ligated into the secretin receptor cDNA.

VeeS

A 74-base pair oligonucleotide was designed as a reverse primer carrying the sequence of the first extracellular loop of the wild type VIP receptor and including an RcaI site downstream of the mutagenized region. PCR was performed using the previously generated VeS chimera as a template. The PCR product was ligated into the HindIII and RcaI sites of the wild type secretin receptor construct.

VeS

This chimeric construct was produced in the same way as VeeS. The wild type secretin receptor construct was used as a template instead of VeS for PCR mutagenesis.

SeeV

Overlap extension PCR (14) was used to construct this chimera, replacing VIP receptor codons 168-183 with secretin receptor codons 175-191.

SeV

The part of SeeV downstream of the BstXI site was excised and ligated into the BstXI and NotI sites of the wild type VIP receptor construct.

All PCR reactions were performed with Taq DNA polymerase in a thermocycler running 35 cycles: 94 °C for 1 min, 55 °C for 2 min, and 72 °C for 3 min. Products were separated on 1% agarose gels and purified on Qiaex resin. Transformations of constructs in pcDNAI/Neo and pcDM8 were performed in MC1061-P3 cells, whereas those in pBK-CMV were performed in XLI-Blue MRF`. Correct sequences of all constructs were confirmed by DNA sequencing using the dideoxynucleotide chain termination method(15) .

Receptor Expression

Constructs were expressed in COS-7 cells maintained in culture in Dulbecco's modified Eagle's medium with 5% fetal clone 2. Cells were transfected with 2-4 µg of DNA using a modification of the DEAE-dextran method, which included MeSO shock and treatment with 0.1 mM chloroquine diphosphate (16). Transfected cells were harvested after 72 h.

Biological Activity Studies

Intracellular cAMP levels were assayed with a [H]cAMP assay kit from Diagnostic Products Corporation (Los Angeles, CA). COS cells were harvested mechanically 72 h after transfection. These were washed with phosphate-buffered saline, resuspended in Krebs-Ringer-HEPES medium incorporating 25 mM HEPES, pH 7.4, 104 mM NaCl, 5 mM KCl, 1.2 mM MgSO, 2 mM CaCl, 1 mM KHPO, 0.2% bovine serum albumin, 0.01% soybean trypsin inhibitor, and 1 mM 3-isobutyl-1-methylxanthine. Hormonal stimulation was performed for 10 min at 37 °C, and the reaction was stopped with ice-cold 6% perchloric acid. The pH was adjusted to 6 with KHCO, and lysates were cleared by centrifugation at 2000 rpm for 10 min.

The generation of standard curves and the measurement of cAMP levels in supernatants of cell lysates were performed as per the manufacturer's instructions. Radioactivity was quantified by scintillation counting in a Beckman LS6000. All assays were performed in duplicate and repeated at least 3 times.

Ligand Binding Studies

Binding studies were performed with enriched plasma membranes prepared from COS cells 72 h after transfection, as we previously described(11) . The secretin analogue, [I-Tyr,pNO-Phe]rat secretin 27, was synthesized and radioiodinated as we described(11) . VIP was radioiodinated oxidatively and purified by high pressure liquid chromatography(17) .

Receptor-bearing membrane preparations (1-10 µg) were incubated with a constant amount of radioiodinated peptide (3-5 pM VIP or secretin analogue(11) ) and increasing concentrations of non-radiolabeled peptide (0-1 µM) for 1 h at room temperature in Krebs-Ringer-HEPES medium. Bound and free radioligand were separated using a Skatron cell harvester with glass fiber filter mats that had been soaked in 0.3% polybrene with bound radioactivity quantified in a -counter. Non-specific binding, assessed in the presence of excess unlabeled analogous peptide (1 µM secretin or VIP), represented less than 2.9 ± 0.4%, with total binding representing 10 ± 1% of radioligand in the incubation.

Statistical Analysis

All observations were repeated at least three times in independent experiments and are expressed as means ± S.E. Differences were determined by using the Mann-Whitney non-parametric test for unpaired values with p < 0.05 considered to be significant.


RESULTS

Biological Activity

Both wild type receptors showed a selectivity for their native agonist ligands of 2 or more orders of magnitude (Fig. 1). The secretin receptor was activated by secretin with an EC of 1.5 nM and by VIP with an EC of 127 nM. The VIP receptor was activated by VIP with an EC of 1.0 nM and by secretin with an EC of 273 nM. Biological activity data for chimeric constructs are also shown in Fig. 1. These data are further summarized in Fig. 2, in which the responsiveness to each hormone was used to organize the data.


Figure 1: Biological responses of wild type and chimeric receptors. Shown are cAMP responses to secretin () and VIP () in the wild type and chimeric secretin and VIP receptors. Values are expressed as the means ± S.E. of at least three independent experiments with data normalized relative to the maximal response to one of the natural agonists. In all constructs, this represented an increase of 2.4 ± 0.1 times the basal levels.




Figure 2: Cyclic AMP responses listed in the order of responsiveness to each hormone. Shown are the means ± S.E. of the concentrations of secretin and VIP that stimulated half-maximal biological responses in at least three independent experiments. A, biological activity ordered according to the responsiveness of chimeras to secretin; B, biological activity ordered according to the responsiveness of chimeras to VIP.



Replacing the amino terminus of the secretin receptor with the same domain of the VIP receptor (VeS) resulted in a construct that changed its specificity from that of the secretin receptor to that of the VIP receptor. This construct was activated by secretin with an EC of 120 nM and by VIP with an EC of 1.7 nM, representing a 100-fold decrease in responsiveness to secretin and a similar increase in responsiveness to VIP. This responsiveness to VIP was not statistically different from the responsiveness of the wild type VIP receptor (p = 0.38), and it had a selectivity of 2 orders of magnitude between VIP and secretin responsiveness like the wild type receptor. This result strongly suggested that the extracellular amino terminus plays a critical role for receptor activation by its natural ligand.

The converse chimera, the VIP receptor with the amino-terminal domain of the secretin receptor (SeV), did not, however, exhibit analogous behavior. Although its maximal increase in cAMP was comparable with that of other constructs, its responsiveness to VIP was reduced 50-fold (EC = 50 nM) while gaining only partial responsiveness to secretin (EC = 30 nM) (p < 0.0001). This construct responded to VIP similarly to the wild type secretin receptor (p = 0.12), further supporting the importance of the amino terminus of the VIP receptor for VIP responsiveness.

In searching for additional receptor domains necessary for secretin responsiveness, a chimera was generated that possessed the secretin receptor sequence for the extracellular amino terminus as well as the first extracellular loop, with the rest of the receptor corresponding to the VIP receptor sequence. The additional replacement of the first extracellular loop by the analogous secretin receptor sequence provided full responsiveness to secretin (EC = 2 nM) (p = 0.37). Interestingly, this chimera was also activated by VIP, with an EC of 47 nM, demonstrating similar responsiveness to VIP to that of the wild type secretin receptor (p = 0.15).

The first extracellular loop of the secretin receptor did not by itself provide a substantial change in the selectivity of the VIP receptor (SeV). Like the chimera with only the amino terminus replaced (SeV), the secretin responsiveness was slightly better (EC = 47 nM) than that of the wild type VIP receptor (p = 0.058). Because this construct still possessed the amino terminus of the wild type VIP receptor, it was very responsive to VIP (EC = 0.5 nM).

The secretin receptor with the first extracellular loop replaced with the corresponding sequence of the VIP receptor responded maximally only to secretin (within the range of concentrations used). The EC for secretin was 4 nM, whereas the EC for VIP was greater than 500 nM.

Ligand Binding

Receptor binding affinity was determined by homologous competition whenever possible. For low affinity ligands, this was not possible and was confirmed by using such a ligand to compete for binding of a higher affinity radioligand. Fig. 3illustrates the competition binding data for the parent wild type receptor. At the wild type secretin receptor, competition for secretin radioligand binding with secretin (IC = 2.2 nM) and VIP (IC > 1 µM) suggested relative binding affinities that correlated with cAMP responses to these hormones. VIP radioligand binding to the wild type VIP receptor yielded analogous data with VIP having an IC of 2.2 nM and secretin having an IC greater than 1 µM.


Figure 3: Binding characteristics of wild type receptors. Shown are results from competition binding experiments utilizing enriched plasma membranes from COS cells transfected with wild type receptor constructs and the noted radioligands. Values are expressed as the means ± S.E. of at least three independent experiments with data representing saturable binding relative to the control condition in the absence of competitor.



Although direct binding of the secretin radioligand to the wild type VIP receptor confirmed its apparent low affinity, the binding of the VIP radioligand to the wild type secretin receptor yielded unexpected results. Homologous competition for that radioligand demonstrated high affinity binding with an IC of 0.6 nM (Fig. 3); however, this binding did not correlate with cAMP generation. VIP-stimulated cAMP generation correlated better with the low affinity binding that was observed when VIP competed for secretin radioligand binding. This suggests that there is a separate and distinct non-biologically relevant high affinity VIP-binding site on the rat secretin receptor. Studies are underway to determine if this is an intrinsic property of the secretin receptor from other species or if this is a unique property of the rat receptor.

Binding data for all constructs are summarized in Fig. 4. All receptors that had demonstrated biological responses to nanomolar concentrations of secretin bound secretin with high affinity (IC = 0.2-2.2 nM), as well as binding VIP with high affinity (IC = 0.6-1.4 nM). All constructs that demonstrated selectivity for biological responses that was similar to that of the wild type VIP receptor displayed high affinity binding of VIP (IC = 1.0-9.3 nM) and low affinity or absent binding of secretin. The SeV construct that showed no selectivity for biological responses to either hormone did not bind either secretin or VIP radioligand with adequate affinity to interpret binding data.


Figure 4: Summary of competition binding data. Shown are the means ± S.E. of the concentrations of secretin and VIP that inhibited half of the saturable ligand binding of the noted radioligand in at least three independent experiments ordered according to the responsiveness to secretin.



Competition binding curves for key chimeric constructs are illustrated in Fig. 5. The VeS construct bound VIP with high affinity. The SeeV construct, like the wild type secretin receptor, bound both secretin and VIP with high affinity.


Figure 5: Binding characteristics of chimeric constructs. Shown are the results from competition binding experiments utilizing enriched plasma membranes from the COS cells transfected with chimeric constructs. All curves represent homologous competition of the same radioligand and cold peptide noted. Values are expressed as the means ± S.E. of at least three independent experiments with data representing saturable binding relative to the control condition in the absence of competitor.




DISCUSSION

General rules have not yet evolved for mechanisms of binding and activation of receptors in the recently described calcitonin-parathyroid hormone family that have as natural agonists peptide ligands with moderately large pharmacophoric domains. In this work, we have utilized a chimeric receptor approach to focus primarily on the potential role of the amino terminus in agonist binding and biological responsiveness. We have chosen the secretin and VIP receptors, which are structurally quite similar yet have distinct agonist specificities. This complements the deletion mutagenesis approach recently utilized for the parathyroid hormone receptor(8, 9) .

The amino terminus is a particularly interesting domain in this family, being quite large (approximately 150 residues) and cysteine-rich. This family of receptors has 8 conserved Cys residues in predicted ectodomains, with 6 of these in the amino terminus. Further evidence for the importance of this domain is a naturally occurring mutation in the growth hormone-releasing hormone receptor. A mis-sense mutation changing Asp-60 (conserved throughout the family) to Gly is reported to disrupt receptor function in the Dwarf Little Mouse(18) .

By replacing the amino terminus of the secretin receptor with the corresponding sequence of the VIP receptor (VeS), a chimera was generated that behaved like the VIP receptor. It responded to VIP with a similar EC to that of the wild type VIP receptor (EC = 1.7 nMversus EC = 1.0 nM) (p = 0.38). Additional receptor domains will likely also contribute to binding and activation of this receptor. It is possible that such domains are sufficiently provided by homologous or identical residues within the secretin receptor sequence in this chimeric construct.

The pattern of secretin-specific determinants on the receptor are different than the VIP-specific determinants. The reciprocal chimeric construct in which the amino terminus of the secretin receptor (SeV) replaced this domain in the VIP receptor displayed only a slight improvement in its secretin responsiveness (p = 0.041). The shift in EC from 273 nM secretin for the wild type VIP receptor to 30 nM secretin for this chimeric construct is consistent with the amino terminus contributing to receptor activation but clearly does not account for full responsiveness (p < 0.0001).

The first extracellular loop of the secretin receptor (SeV) also contributed a small amount to the secretin responsiveness of the VIP receptor. Adding both the amino terminus and the first extracellular loop of the secretin receptor together (SeeV) resulted in a marked effect in the secretin responsiveness of the VIP receptor, being similar to that of the wild type secretin receptor (p = 0.37). Of interest, the VIP responsiveness of this construct decreased from that of the VIP receptor to that of the secretin receptor, consistent with the critical role of the amino terminus in VIP effects. As expected, such an effect was not seen when only the first extracellular loop was replaced in the VIP receptor.

The VeeS chimera representing the secretin receptor with the extracellular amino terminus and the first extracellular loop of the VIP receptor further confirmed this evolving theme. The amino terminus is critical for receptor activation by VIP, and the first extracellular loop did not contribute further to distinguishing VIP from secretin.

Organizing the chimeras according to their responsiveness to secretin and VIP facilitates envisioning the distribution of determinants critical for receptor activation by the respective ligands (Fig. 2). Three groups can be distinguished based on secretin responsiveness: the wild type secretin receptor and the SeeV and VeS chimeras responded well to secretin; the SeV and SeV chimeras showed intermediate responses to secretin; and VeS, VeeS, and the wild type VIP receptor showed poor responses to secretin. Based on the responsiveness to VIP, again three groups could be distinguished. The wild type VIP receptor and the SeV, VeS, and VeeS chimeras responded well to VIP; SeV and SeeV showed intermediate responses; and VeS and the wild type secretin receptor responded poorly.

Wild type secretin and VIP receptors exhibit sensitive responses to their natural ligands, with selectivities of at least 2 orders of magnitude. For both receptors, the presence of the amino terminus is necessary for the activation of the receptors by low concentrations of native agonists. Although this domain of the VIP receptor can alone account for a selectivity of 2 orders of magnitude, in the secretin receptor additional determinants are necessary to activate the receptor with physiological concentrations and high selectivity. Further addition of the first extracellular loop of the secretin receptor can provide full responsiveness to secretin.

The ligand binding studies nicely complement the biological activity studies. These showed high affinity binding of secretin to the wild type secretin receptor and the SeeV and VeS chimeras, correlating with the sensitive biological responses to secretin. All other constructs and the wild type VIP receptor bound secretin poorly. The correlation between high affinity secretin binding and biological responses might suggest that the determinants critical for receptor binding also mediate activation. However, a more detailed survey will likely reveal distinct residues that will distinguish between contributions to ligand binding and receptor activation. Secretin-binding determinants are clearly present in the extracellular amino terminus and in the first extracellular loop.

In contrast, VIP bound with high affinity to both wild type VIP and secretin receptors and to almost all chimeric constructs. However, this binding only resulted in a biological response at VIP-like receptors. At the secretin receptor, the cAMP response generated by VIP correlated with its low affinity binding to the site that bound the secretin radioligand. This is a novel observation that was previously not possible with receptors on native cells in which more than one receptor might have been present. It was previously felt that there were distinct ``secretin-preferring'' receptors and ``VIP-preferring'' receptors(19) . Whereas distinct wild type secretin and VIP receptors do exist and may coexist on a single native cell, binding data for the recombinant wild type rat secretin receptor in a system in which we can be certain that a VIP receptor does not exist demonstrate both types of binding. Biological activities, however, clearly distinguish between these. It will be of interest to determine if high affinity binding of VIP is a property of all secretin receptors or if this is peculiar to the rat receptor.

In this study we have demonstrated that the amino terminus of secretin and VIP receptors with their characteristic structural features play a critical role for the specificity of binding affinity and receptor activation. For the secretin receptor, other extracellular sites are critical as well. Our results provide a basis for future studies defining the specific determinants for affinity and specificity of these receptors. Although chimeric constructs substituting smaller portions of these receptors may localize determinants more precisely, roles of distinct amino acid residues will also need to be addressed.

  
Table: Secretin-VIP receptor chimeras

The numbers in parentheses represent the amino acid codons in the wild type receptor proteins.



FOOTNOTES

*
This work was supported by National Institutes of Health Grant DK46577 (to L. J. M.) and a grant from the Studienstiftung Des Deutschen Volkes (to M. H. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Center for Basic Research in Digestive Diseases, Guggenheim 17, Mayo Clinic, Rochester, MN 55905. Tel.: 507-284-0680.

The abbreviations used are: VIP, vasoactive intestinal polypeptide; PCR, polymerase chain reaction.


ACKNOWLEDGEMENTS

We acknowledge the excellent technical assistance of E. Holicky, D. Pinon, and I. Ferber and the excellent secretarial assistance of S. Erickson.


REFERENCES
  1. Lin, H. Y., Harris, T. L., Flannery, M. S., Aruffo, A., Kaji, E. H., Gorn, A., Kolakowski, L. F., Jr., Lodish, H. F., and Goldring, S. R.(1991) Science254, 1022-1026 [Medline] [Order article via Infotrieve]
  2. Jüppner, H., Abou-Samra, A., Freeman, M., Kong, X. F., Schipani, E., Richards, J., Kolakowski, L. F., Hock, J., Potts, J. T., Kronenberg, H. M., and Segre, G. V.(1991) Science254, 1024-1026 [Medline] [Order article via Infotrieve]
  3. Segre, G. V., and Goldring, S. R.(1993) Trends Endocrinol. Metab.4, 309-314
  4. Mutt, V.(1980) in Gastrointestinal Hormones (Glass, G. B. J., ed) pp. 85-126, Raven Press, New York
  5. Schwartz, T. W.(1994) Curr. Opin. Biotechnol.5, 434-444 [Medline] [Order article via Infotrieve]
  6. Coughlin, S. R.(1994) Curr. Opin. Cell Biol.6, 191-197 [Medline] [Order article via Infotrieve]
  7. Huang, R.-R. C., Yu, H., Strader, C. D., and Fong, T. M.(1994) Mol. Pharmacol.45, 690-695 [Abstract]
  8. Lee, C., Gardella, T. J., Abou-Samra, A.-B., Nussbaum, S. R., Segre, G. V., Potts, J. T., Jr., Kronenberg, H. M., and Jüppner, H.(1994) Endocrinology135, 1488-1495 [Abstract]
  9. Jüppner, H., Schipani, E., Bringhurst, F. R., McClure, I., Keutmann, H. T., Potts, J. T., Jr., Kronenberg, H. M., Abou-Samra, A. B., Segre, G. V., and Gardella, T. J.(1994) Endocrinology134, 879-884 [Abstract]
  10. Ishihara, T., Nakamura, S., Kaziro, Y., Takahashi, T., Takahashi, K., and Nagata, S.(1991) EMBO J.10, 1635-1641 [Abstract]
  11. Ulrich, C. D., II, Pinon, D. I., Hadac, E. M., Holicky, E. L., Chang-Miller, A., Gates, L. K., and Miller, L. J.(1993) Gastroenterology105, 1534-1543 [Medline] [Order article via Infotrieve]
  12. Ishihara, T., Shigemoto, R., Mori, K., Takahashi, K., and Nagata, S. (1992) Neuron8, 811-819 [Medline] [Order article via Infotrieve]
  13. Rosselin, G.(1986) Peptides (Elmsford) 7, 89-100 [Medline] [Order article via Infotrieve]
  14. Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K., and Pease, L. R. (1989) Gene (Amst.) 77, 61-68 [CrossRef][Medline] [Order article via Infotrieve]
  15. Sanger, F., Nicklen, S., and Coulson, A. R.(1977) Proc. Natl. Acad. Sci. U. S. A.74, 5463-5467 [Abstract]
  16. Lopata, M. A., Cleveland, D. W., and Sollner-Webb, B.(1984) Nucleic Acids Res.12, 5707-5717 [Abstract]
  17. Hunter, W. M., and Greenwood, F. C.(1962) Nature194, 495-496
  18. Godfrey, P., Rahal, J. O., Beamer, W. G., Copeland, N. G., Jenkins, N. A., and Mayo, K. E.(1993) Nat. Genet.4, 227-232 [Medline] [Order article via Infotrieve]
  19. Bissonnette, B. M., Collen, M. J., Adachi, H., Jensen, R. T., and Gardner, J. D.(1984) Am. J. Physiol.246, G710-G717

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.