©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mapping of an NH-terminal Ligand Binding Site of the Insulin Receptor by Alanine Scanning Mutagenesis (*)

(Received for publication, November 4, 1994; and in revised form, December 6, 1994)

Paul F. Williams (1)(§)(¶) Dennis C. Mynarcik (1)(¶) Gui Qin Yu (1) Jonathan Whittaker (1) (2)(**)

From the  (1)Department of Medicine and (2)Physiology and Biophysics, State University of New York, Stony Brook, New York 11794

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Affinity labeling studies and mutational analyses have implicated the involvement of a predicted domain of the insulin receptor (L1, amino acids 1-119) in ligand binding. In order to obtain a higher resolution localization of this ligand binding site, we have performed alanine scanning mutagenesis of this domain. Alanine mutant cDNAs encoding a secreted recombinant insulin receptor extracellular domain were expressed transiently in adenovirus transformed human embryonic kidney cells and the affinity of the expressed receptor for insulin was determined. Mutation of 14 amino acids located in four discontinuous peptide segments to alanine was disruptive of insulin binding: Segment 1, amino acids 12-15; Segment 2, amino acids 34-44; Segment 3, amino acids 64-67; and Segment 4, amino acids 89-91. The quantitative contribution of the four segments to the free energy of insulin binding was 1 > 3 > 2 > 4. Of the 14 amino acids whose mutation compromised insulin binding, 3 are charged, 3 hydrophobic, 5 aromatic, and 3 are amides.


INTRODUCTION

Insulin initiates signal transduction in target cells by binding to a specific cell surface receptor(1) . This probably leads to conformational changes in the extracellular domains, which are transmitted across the cell membrane and result in activation of the receptor's tyrosine kinase activity. The molecular details of these events are obscure and will require a detailed understanding of the structure function relationships of the protein, in particular those of the extracellular domain.

The primary structure of this region has been deduced from the predicted amino acid sequence of the cloned human insulin receptor cDNA (2, 3) . It is composed of two disulfide-linked heterodimers, each of which is composed in turn of an M(r) 135,000 alpha subunit, which is entirely extracellular, linked by a disulfide bond to an M(r) 95,000 beta subunit, which has an extracellular domain, a single alpha helical transmembrane domain, and an intracellular domain containing the tyrosine kinase catalytic activity. The alpha subunit contains a cysteine-rich domain homologous to that of the epidermal growth factor receptor, and there are also possibly two fibronectin type III repeats in the extracellular domain(4) .

Bajaj et al.(5) have proposed a hypothetical model of the tertiary structure of the receptor extracellular domain based on homologies between the primary structures of the epidermal growth factor and insulin receptor families of tyrosine kinases(5) . This model predicts that there are two homologous globular domains flanking the cysteine domains: domain L1 containing amino acids 1-119 and domain L2 containing amino acids 311-428. Each contains repeating structural motifs (Motifs I-V) composed of alpha helix, beta turn beta, and hypervariable structures. Since all deletions and insertions occur in the hypervariable structures in the sequence alignments obtained for these proteins with this model, it was suggested that these may represent components of ligand binding domains.

For the insulin receptor this proposal has received support from recent experimental observations. Affinity labeling studies and mutagenic analyses suggest the involvement of both the NH(2) terminus of the molecule (6, 7) and also a region COOH-terminal to the cysteine domain (8) in insulin binding. Furthermore, studies of the ligand properties of chimeric receptors produced from the insulin and the related IGF-1 receptors indicate that residues 1-68 (9) and 325-524 (10) are involved in conferring ligand specificity, although the role of the latter residues is probably minor. In addition point mutations of amino acids located within the L1 domain, Asn-15 (11) and Phe-89(12, 13) , have been shown to compromise high affinity insulin binding.

In view of this compelling evidence for the involvement of the L1 domain of the insulin receptor in ligand binding, we have undertaken, in the present study, a high resolution analysis of the residues involved by alanine scanning mutagenesis as a first step in elucidating the detailed mechanism of insulin receptor signal transduction. Since studies of insulin structure function relationships suggest a prominent role for aromatic residues in receptor interactions(14) , we performed both aromatic to alanine and charged to alanine mutagenesis. The results of these experiments indicate that the L1 domain of the insulin receptor contains an insulin binding site composed of four discontinuous polypeptide segments containing 14 amino acids, the mutation of which compromised insulin binding.


MATERIALS AND METHODS

General Procedures

All molecular biological procedures, including agarose gel electrophoresis, restriction enzyme digestion, ligation, bacterial transformation, and DNA sequencing, were performed by standard methods(15) . All oligonucleotides were synthesized on an Applied Biosystems model 391A PCR Mate synthesizer. Restriction and DNA modifying enzymes were from New England Biolabs (Beverly, MA). Porcine insulin was purchased from Bios Pacific (Emeryville, CA), and insulin radioiodinated at tyrosine A-14 of the A chain I-[Tyr-A14]insulin (^1)was from Amersham Corp. Protease inhibitors were from Boehringer Mannheim. 293 EBNA cells were purchased from InVitrogen (San Diego, CA). Media for tissue culture was from Mediatech (Herndon, VA) and serum from Sigma. Anti-insulin receptor monoclonal antibody 18-44 (16) was generously provided by Drs. M. Soos and K. Siddle (University of Cambridge, United Kingdom).

Oligonucleotide-directed Mutagenesis

Oligonucleotide-directed mutagenesis was performed by the method of Kunkel(17) . A 1.9-kilobase pair fragment of the human insulin receptor cDNA extending from a Kozak consensus initiation sequence preceded by a HindIII restriction site to the BamHI restriction site (18) subcloned into the phagemid pTZ18U was used as the template for mutagenesis. Where possible, restriction sites were deleted or introduced with the specific mutation in order to permit enrichment of mutants by restriction selection or purification and to facilitate the screening of mutants.

Expression of Mutant Insulin Receptor cDNAs

The mutant insulin receptor cDNAs encoding a secreted receptor extracellular domain were reconstructed in the expression vector pCDE. This a modification of pCEP4 (InVitrogen) from which the coding region for the EBV nuclear antigen has been deleted; thus, this vector contains the cytomegalovirus immediate early promoter for the expression of cloned cDNAs, the EBV origin of replication for episomal replication in cells expressing EBV nuclear antigen, and a hygromycin resistance gene for the selection of stable cell lines.

These constructs were expressed in 293EBNA cells (an adenovirus transformed human kidney cell line expressing EBV nuclear antigen) by transfection with 2 µg of Miniprep DNA using the commercially available lipofection reagent Lipofectamine (Life Technologies) according to the manufacturers' directions. For analysis of transient expression, media and cells were harvested 1 week after transfection. Conditioned medium was concentrated prior to assay using Centriprep 100 centrifugal concentrators (Amicon, Beverly, MA).

We chose to utilize the extracellular domain for these experiments, as it is expressed in large amounts by this expression system, and insulin only binds to a single homogeneous population of binding sites in this protein which have been shown to be contained within the L1 domain(9) , thus simplifying the analysis of binding data.

Insulin Binding Assays

Soluble insulin receptor binding assays were performed using a microtiter plate antibody capture assay. Microtiter plates (Immulon 4, Dynal Inc., Lake Success, NY) were incubated with affinity purified goat anti-mouse IgG (50 µl/well of 20 µg/ml solution in 0.2 M sodium carbonate, pH 9.4) for 2 h at room temperature. After washing and blocking for 15 min with 250 µl of SuperBlock(TM) (Pierce), wells were then incubated overnight at 4 °C with a 1:100 dilution of crude ascites of anti-insulin receptor monoclonal antibody 18-44 in SuperBlock(TM). After washing with phosphate-buffered saline, wells were incubated for 4h at 4 °C with soluble receptor, diluted to give 10-20% I-[Tyr-A14]insulin binding in the absence of added unlabeled insulin under assay conditions. After washing with wash buffer (0.15 M sodium chloride, 20 mM Hepes, pH 7.8, 1% bovine serum albumin (w/v), 0.1% Triton X-100 (v/v), 0.1% Tween 20 (v/v), and 0.02% sodium azide (w/v)), wells were incubated overnight at 4 °C with I-[Tyr-A14]insulin (50 pM) and varying concentrations of unlabeled insulin, in 100 µl of binding buffer. To terminate the assay, wells were aspirated and washed three times with 300 µl of ice-cold wash buffer and then counted.

Insulin binding data were analyzed by the LIGAND program (20) in order to obtain the K(d) of the expressed protein. Trasfection and binding assays were repeated at least once to confirm the K(d) of each mutant. Each result is the mean of two experiments.

Immunoblotting

Immunoblotting of the insulin receptor alpha subunit in conditioned medium and detergent lysates of transfected cells was performed according to standard methods using a commercially available antibody directed toward the C terminus of the alpha subunit (Upstate Biotechnology, Inc., Lake Placid, NY). Blots were visualized by enhanced chemiluminescence (ECL, Amersham).


RESULTS

As described previously, insulin binding to recombinant insulin receptor extracellular domain secreted by transiently transfected 293EBNA cells displayed simple kinetics with a linear Scatchard plot (data not shown)(19) . Analysis with the LIGAND program (20) indicated a single population of binding sites with a K(d) of 1.41 ± 0.09 times 10M (mean ± S.E., n = 6). Since previous studies utilizing alanine scanning mutagenesis have demonstrated that meaningful changes in affinity produced by a single alanine substitution range from 2- to 100-fold (21) , in the experiments described below we regarded any mutant with a greater than 2-fold increase in K(d), i.e.K(d) greater than 2.8 times 10M, as causing a significant disruption in insulin-receptor interactions.

The initial mutants analyzed were clustered charged/aromatic mutations. The results of these analyses are shown in Table 1, part A. None of these mutants displayed any abnormality except the the receptor with the simultaneous mutation of Phe-64 and Arg-65 (A64F/A65R), which did not appear to be secreted although Western blotting of lysates of the transfected cells revealed detectable levels of receptor precursor (data not shown), a pattern of disruption of post-translational processing indicative of impaired folding of the nascent proreceptor(11, 22) . Mutations of Phe-64 and Arg-65 were therefore analyzed individually (see below).



We then proceeded to perform alanine scanning mutagenesis of individual charged amino acids (Table 1, part B). This identified Arg-14 as a major determinant of insulin binding; mutation of this residue to alanine led to the expression of a receptor whose affinity for insulin was too low to be measured by the methodology used for this study. In the same region mutation of D12 to alanine produced a 5-fold reduction in affinity for insulin (K(d) = 9.12 times 10M). A further mutation of Glu-44 to alanine caused a 3-4 fold decrease in affinity (K(d) = 6.82 times 10M). Mutation of Arg-65 to alanine had no effect on expression or secretion of the resulting protein, and its affinity for insulin was normal. Mutation of Glu97 resulted in impaired receptor secretion, although the precursor of this mutant protein was detectable by immunoblotting of lysates of transfected cells (data not shown), again suggesting malfolding of the mutant.

We next undertook aromatic to alanine mutagenesis (Table 1, part C). This revealed 4 additional residues whose mutation significantly compromised insulin binding; Phe-39 (K(d) = 35.70 times 10M), Phe-64 (affinity too low to be accurately measured), Phe-89 (K(d) = 5.17 times 10M), and Tyr-91 (K(d) = 3.85 times 10M). Phe-39 is located in a region that has previously been reported to be important for conferring insulin specificity in studies of the binding properties of chimeric insulin-IGF-1 receptors(9) . Phe-89 has also previously been reported to be important for high affinity insulin binding(12, 13) . Mutation of Tyr-60 and Phe-96 led to a failure of secretion into the medium although the presence of their precursors were visualized in cell lysates by Western blotting (data not shown), suggesting that these mutations compromise the structure of the receptor. It is of interest that Phe-96 is a neighbor of Glu-97 whose mutation to alanine also caused impaired expression in the charged to alanine mutagenesis experiments, suggesting the importance of this region for folding of the molecule into a native conformation. These residues are conserved in the insulin receptor, the IGF-1 receptor, and the insulin receptor-related protein(23) , further emphasizing their critical roles in the maintenance of the conformation of this family of proteins.

To further define the structures in which alanine mutations compromised binding, we mutated amino acids surrounding those that we had previously identified as being necessary for high affinity binding. To avoid potential structural perturbations, we did not mutate prolines, cysteines, or potential N-linked glycosylation sites. The results of these scans confirmed that the insulin binding epitope was composed of four discontinuous peptide segments; Segment 1 from Asp-12 to Asn-15, Segment 2 from Gln-34 to Glu-44, Segment 3 from Phe-64 to Tyr-67, and Segment 4 from Phe-89 to Tyr-91 (see Table 1, part D, and Fig. 1). In Segment 1, mutations produced decreases in affinity ranging from 6- to 7-fold (D12A) to too low to accurately determine (R14A).In the the second segment, mutations produced 3-fold (M38A) to 25-fold (F39A) reductions in affinity. In addition, mutations L33A, I35A, and L37A led to a failure of secretion of protein, although expression of precursor is detectable in cell lysates (data not shown). Mutations K40A, T41A, and R42A were without effect on insulin binding. In Segment 3 only mutations F64A and Y67A reduced affinity for insulin (too low to be determined and 2.2-fold, respectively). Mutations R65A and V66A produced receptors with normal affinity for insulin. In Segment 4 mutations reduced affinity from 2.5-fold (Y91A) to 6-fold (N90A).


Figure 1: Alanine scanning mutagenesis of the NH(2)-terminal ligand binding site of the recombinant secreted insulin receptor. Data from Table 1are expressed as a ratio of the K of the mutant to that of wild type recombinant insulin receptor (K(mut)/K(wt)). Results for amino acids 1-60 are shown in the upperpanel and those for amino acids 61-120 in the lowerpanel. Amino acids are designated by single-letter code. The K(mut)/K(wt) could not be accurately determined for mutations of Arg-14 and Phe-64 (designated by *). K(mut)/K(wt) for the mutation of N15 is 250 as indicated on the figure.




DISCUSSION

In the present study we have identified 14 amino acids organized into four discontinuous segments, which appear to be the major functional determinants of the N-terminal ligand binding domain of the insulin receptor - Segment 1 (Asp-12, Ile-13, Arg-14, and Asn-15), Segment 2 (Gln-34, Leu-36, Met-38, Phe-39, and Glu-44), Segment 3 (Phe-64 and Tyr-67), and Segment 4 (Phe-89, Asn-90, and Tyr-91). Of the 14 amino acids, 3 are charged, 3 hydrophobic, 5 aromatic, and 3 are amides. The prominence of the aromatic residues further emphasizes the role of aromatic interactions in insulin receptor interactions. The predicted secondary structure of the four segments according to the model of Bajaj et al.(5) is: Segment 1: beta strand, alpha helix (Motifs I and II, respectively); Segment 2: beta strand, loop (Motif II); Segment 3: loop (Motif III); Segment 4: loop (Motif IV).

Thus, as suggested by these authors, the predicted loop structures appear to play a prominent role in ligand binding. However, in contrast to immunoglobulins(24) , the ligand binding epitope is not confined to these structures as both predicted beta strand and alpha helical structures are also involved. Precedents for this have been reported for the growth hormone receptor(21) .

Several lines of evidence suggest that the decreases in affinity observed with these mutations are probably direct effects on ligand receptor interactions rather than the consequences of misfolding of the mutant proteins. Previous analyses of protein structure and function have shown that the effects of alanine mutants tend to be localized and nondisruptive of global protein structure(21) . In the case of the growth hormone-growth hormone receptor interactions, crystallographic studies have confirmed the involvement of determinants identified by scanning mutagenesis in hormone receptor interactions(25) . Second, in common with other membrane and secreted proteins (for review, see (26) ), studies of naturally occurring mutants of the insulin receptor associated with extreme insulin resistance (11) and of secreted COOH-terminal deletion mutants of the receptor (22) indicate that there is a strict requirement for folding into a native conformation prior to completion of post-translational processing and transport to the membrane or secretion. In the present study, all the mutants disruptive of insulin binding were secreted at levels comparable to that of the wild type protein and those of mutants that were without effect on insulin binding.

Of the four segments that we have identified, Segment 1 appears to be quantitatively the most important in its contribution to the free energy of binding, probably followed by Segment 3, and then 2, with the smallest contribution coming from Segment 4. Mutation of Arg-14 to alanine results in a receptor with an unmeasurably low affinity and mutation of Asn-15 to alanine producing a greater than 200-fold decrease. Interestingly a mutation at this position has been identified in a patient with extreme insulin resistance(11) . In this mutant the asparagine was mutated to lysine and resulted in impaired folding of the receptor and retention in the endoplasmic reticulum. However, those receptors that did reach the cell membrane exhibited a 5-fold decrease in affinity for insulin. In contrast the substitution of alanine results in the expression of a protein that appears to have native structure, since it is secreted in quantities comparable to that of the wild type receptor and post-translational processing does not appear to be impaired. The alanine substitution, however, produced a much more profound decrease in affinity than the lysine substitution, presumably because of the loss of the side chain. Phe-64 in segment 3 also appears to make a contribution to the free energy of binding comparable to that of Arg-14. However, accurate determination of the affinity of these residues will be required before we can definitively evaluate their relative contributions to the free energy of insulin binding.

Studies of insulin and IGF-1 specificity with chimeric receptors have identified amino acids 1-68 as being the major determinant of insulin specificity of the secreted soluble receptor(9) . Within this region it appears that amino acid differences in the region 38-68 are the most important. Thus, on the basis of the results of our scanning mutagenesis, one would predict Met-38, Phe-39, and Tyr-67 to be the major determinants of specificity with the major energetic contribution being provided by Phe-39. Certainly the combined effects of mutation of these residues to alanine are consistent with the observed differences in affinities between the secreted recombinant insulin and IGF-1 receptors for insulin (9) (assuming that resulting free energy changes of the combined mutations are additive; (27) ). The data acquired from scanning mutagenesis also allow us to predict the residues in the insulin receptor-related protein that contribute to the low affinity of this protein for insulin. There are significant sequence differences between the two proteins in Segments 1 and 4(23) . In Segment 1, Asn-15 of the insulin receptor is replaced by serine in the insulin receptor related protein; and in epitope 4, Phe-89 and Asn-90 of the insulin receptor are replaced by leucine and glycine, respectively. The combination of these substitutions is likely to cause a profound decrease in affinity for insulin. Experiments are currently in progress to test these predictions.

While it should be noted that these results were obtained with a recombinant receptor that has an affinity at least an order of magnitude lower than that of wild type receptor(28) , there are good reasons to believe that the results can be extrapolated to the native receptor. Recently two models have been proposed to explain the complex kinetics of insulin binding(28, 29) . Both propose that there are two distinct receptor binding sites on the insulin molecule and that there are two distinct insulin binding sites on each alpha subunit, Site 1 and Site 2. Insulin first binds to the site with higher affinity, Site 1, on one alpha subunit and then cross-links the two heterodimers by binding to Site 2 on the second alpha subunit, generating the high affinity component of the receptor interaction. Binding of a second insulin molecule in this manner disrupts the cross-linking of the first and accelerates its dissociation (negative cooperativity). In the recombinant secreted receptor and the isolated heterodimer, insulin only binds to Site 1, and hence this interaction displays a lower affinity than that of the native receptor and simple binding kinetics. From the data presented by Shaffer, it is apparent that the affinity of Site 1 is very much greater than that of Site 2. Thus it would be expected mutations producing changes in the affinity of this binding site for insulin, in the recombinant secreted receptor would also produce comparable changes in affinity of the holoreceptor. This conclusion is supported by experimental evidence for Segments 2-4. It has been reported that the relative increase in affinity for insulin of chimeric IGF-1 receptors in which amino acids 1-62 have been substituted by the corresponding region of the insulin receptor is comparable for both the secreted and full-length recombinant proteins, suggesting that the contributions to the free energy of binding of Met-38, Phe-39, and Tyr-67 that we have demonstrated will be comparable in the both forms of the receptor. Additionally, Schumacher et al.(13) have studied the properties of the F89A mutation in the holoreceptor, and the changes in affinity that they reported are very similar to those we have found for the secreted form of this mutant.

This study thus confirms the utility of alanine scanning mutagenesis for the investigation of insulin-receptor interactions. This approach is has enabled us to map a discontinuous epitope of the insulin receptor involved in insulin binding, which contains polypeptide segments that would have escaped attention using previously employed systematic mutagenic approaches. However, while this analysis implies direct molecular interactions between this epitope and the insulin molecule, it does not provide proof of such interactions. Direct proof will require a high resolution structural analysis.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant DK 42171 (to J. W.). 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.

§
Present address: Dept. of Endocrinology, Blackburn Building, University of Sydney, Sydney, New South Wales 2006, Australia.

These authors contributed equally to the work described in this paper and should be considered joint first authors.

**
To whom correspondence should be addressed: Division of Endocrinology, Dept. of Medicine, Health Sciences Center HSC T15, Rm. 050, SUNY at Stony Brook, Stony Brook, NY 11794-8154. Tel.: 516-444-1036; Fax: 516-444-2493.

(^1)
The abbreviations used are: I-[Tyr-A14]insulin, insulin radioiodinated at tyrosine 14 of the A-polypeptide chain; EBV, Epstein-Barr virus.


REFERENCES

  1. Lee, J. & Pilch, P. F. (1994) Am. J. Physiol. 266, C319-C334
  2. Ebina, Y., Ellis, L., Jarnagin, K., Edery, M., Graf, L., Clauser, E., Ou, J. H., Masiarz, F., Kan, Y.-W., Goldfine, I. D., Roth, R. A. & Rutter, W. J. (1985) Cell 40, 747-758 [Medline] [Order article via Infotrieve]
  3. Ullrich, A., Bell, J. R., Chen, E. Y., Herrera, R., Petruzzelli, L. M., Dull, T. J., Gray, A., Coussens, L., Liao, Y.-C., Tsubokawa, M., Mason, A., Seeburg, P. H., Grunfeld, C. & Rosen, O. M. (1985) Nature 313, 756-761 [Medline] [Order article via Infotrieve]
  4. Schaefer, E. M., Erickson, H. P., Federwisch, M., Wollmer, A. & Ellis, L. (1992) J. Biol. Chem. 267, 23393-23402 [Abstract/Free Full Text]
  5. Bajaj, M., Waterfield, M. D., Schlessinger, J., Taylor, W. R. & Blundell, T. (1987) Biochim. Biophys. Acta 916, 220-226 [Medline] [Order article via Infotrieve]
  6. Wedekind, F., Baer-Pontzen, K., Bala-Mohan, S., Choli, D., Zahn, H. & Brandenburg, D. (1989) Biol. Chem. Hoppe-Seyler 370, 251-258 [Medline] [Order article via Infotrieve]
  7. Waugh, S. M., DiBella, E. E. & Pilch, P. F. (1989) Biochemistry 28, 3448-3455 [Medline] [Order article via Infotrieve]
  8. Fabry, M., Schaefer, E., Ellis, L., Kojro, E., Fahrenholz, F. & Brandenburg, D. (1992) J. Biol. Chem. 267, 8950-8956 [Abstract/Free Full Text]
  9. Andersen, A. S., Kjeldsen, T., Wiberg, F. C., Vissing, H., Schäffer, L., Rasmussen, J. S., De Meyts, P. & Møller, N. P. H. (1992) J. Biol. Chem. 267, 13681-13686 [Abstract/Free Full Text]
  10. Schumacher, R., Soos, M. A., Schlessinger, J., Brandenburg, D., Siddle, K. & Ullrich, A. (1993) J. Biol. Chem. 268, 1087-1094 [Abstract/Free Full Text]
  11. Kadowaki, T., Kadowaki, H., Accili, D. & Taylor, S. I. (1990) J. Biol. Chem. 265, 19143-19150 [Abstract/Free Full Text]
  12. DeMeyts, P., Gu, J. L., Shymko, R. M., Kaplan, B. E., Bell, G. I. & Whittaker, J. (1990) Mol. Endocrinol. 4, 409-416 [Abstract]
  13. Schumacher, R., Mosthaf, L., Schlessinger, J., Brandenburg, D. & Ullrich, A. (1991) J. Biol. Chem. 266, 19288-19295 [Abstract/Free Full Text]
  14. Baker, E. N., Blundell, T. L., Cutfield, J. F., Cutfield, S. M., Dodson, E. J., Dodon, G. G., Crowfoot Hodgkin, D. M., Hubbard, R. E., Isaacs, N. W., Reynolds, C. D., Sakabe, K., Sakabe, N. & Vijayan, N. M. (1988) Phil. Trans. R. Soc. Lond. Ser. B Biol. Sci. 319, 369-456 [Medline] [Order article via Infotrieve]
  15. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  16. Soos, M. A., Siddle, K., Stanley, K. K., Baron, M. D., Heward, J. M., Luzio, J. P., Bellatin, J. & Lennox, E. S. (1986) Biochem. J. 235, 199-208 [Medline] [Order article via Infotrieve]
  17. Kunkel, T. A., Bebenek, K. & McClary, J. (1991) Methods Enzymol. 204, 125-139 [Medline] [Order article via Infotrieve]
  18. Whittaker, J., Okamoto, A. K., Thys, R., Bell, G. I., Steiner, D. F. & Hofmann, C. A. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5237-5241 [Abstract]
  19. Markussen, J., Halstrom, J., Wiberg, F. C. & Schaffer, L. J. (1991) J. Biol. Chem. 266, 18814-18818 [Abstract/Free Full Text]
  20. Munson, P. J. & Rodbard, D. (1980) Anal. Biochem. 107, 220-239 [Medline] [Order article via Infotrieve]
  21. Wells, J. A. (1991) Methods Enzymol. 202, 390-411 [Medline] [Order article via Infotrieve]
  22. Schaefer, E. M., Siddle, K. & Ellis, L. J. (1990) J. Biol. Chem. 265, 13248-13253 [Abstract/Free Full Text]
  23. Shier, P. & Watt, V. M. (1989) J. Biol. Chem. 264, 14605-14608 [Abstract/Free Full Text]
  24. Mariuzza, R. A., Phillips, S. E. & Poljak, R. J. (1987) Annu. Rev. Biophys. Biophys. Chem. 16, 139-159 [CrossRef][Medline] [Order article via Infotrieve]
  25. de Vos, A. M., Ultsch, M. & Kossiakoff, A. A. (1992) Science 255, 306-312 [Medline] [Order article via Infotrieve]
  26. de Silva, A. M., Balch, W. E. & Helenius, A. J. (1990) Cell Biol. 111, 857-866
  27. Wells, J. A. (1990) Biochemistry 37, 8509-8517
  28. Schaffer, L. (1994) Eur. J. Biochem. 221, 1127-1132 [Abstract]
  29. De Meyts, P. (1994) Diabetologia 37,Suppl. 2, S135-S148

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