Kathryn McLane - Academia.edu (original) (raw)

Papers by Kathryn McLane

Research paper thumbnail of Transplantation of a 17-Amino Acid -Helical DNA-Binding Domain Into an Antibody Molecule Confers Sequence-Dependent DNA Recognition

Research paper thumbnail of The Nicotinic Acetylcholine Receptor: Structure and Autoimmune Pathology

Critical Reviews in Biochemistry and Molecular Biology, 1994

ABSTRACT

Research paper thumbnail of anti-TNF antibodies and methods of use

An antibody that can bind TNF alpha, wherein the antibody comprises: (a) a polypeptide comprising... more An antibody that can bind TNF alpha, wherein the antibody comprises: (a) a polypeptide comprising a polypeptide having the sequence shown in SEQ ID NO: 2; and (b) a polypeptide comprising a polypeptide having the sequence shown in SEQ ID NO: 380, wherein said antibody binds to TNF alpha higher than infliximab affinity as determined using a Biacore assay and in the the VH region infliximab has the sequence shown in SEQ ID NO: 1 and the VL of infliximab has the sequence shown in SEQ ID NO: 379.

Research paper thumbnail of Polypeptides de liaison de chaine variable de type immunoglobuline et procedes d'utilisation

Cette invention concerne une population variee isolee de polypeptides de liaison de type VH. Chaq... more Cette invention concerne une population variee isolee de polypeptides de liaison de type VH. Chaque polypeptide de liaison de cette population comprend une combinaison non certifiee d'un polypeptide code par exon de zone VH d'immunoglobuline, d'un polypeptide code par exon de zone JH et d'un polypeptide code par exon de zone D, ces polypeptides codes par exon de zone VH, D et JH sont joints en un seul polypeptide formant un polypeptide de liaison de type immunoglobuline VH, ou un fragment fonctionnel de celui-ci.

Research paper thumbnail of Species- and subtype-specitid recognition by antibody WF6 of a sequence segment forming an α-bungaratoxin binding site on the nictoinic acetylcholine receptor α subunit

Journal of Receptor Research, 1992

Research paper thumbnail of Immunoglobulin-like variable chain binding polypeptides and methods of use

Research paper thumbnail of Anti-TNFα antibodies

Research paper thumbnail of Identification of sequence segments forming the alpha-bungarotoxin binding sites on two nicotinic acetylcholine receptor alpha subunits from the avian brain

Journal of Biological Chemistry, 1991

Research paper thumbnail of Localization of sequence segments forming a kappa-bungarotoxin-binding site on the alpha 3 neuronal nicotinic receptor

Journal of Biological Chemistry, 1990

To identify the sequence segments of the a3 subunit of the neuronal nicotinic acetylcholine recep... more To identify the sequence segments of the a3 subunit of the neuronal nicotinic acetylcholine receptor (N-nAChR) forming the binding site for the cholinergic antagonist K-bungarotoxin (K-BGT), overlapping peptides corresponding to the complete a3 sequence were tested for their ability to bind 12'1-labeled K-BGT. Two peptides located within the N-terminal extracellular domain specifically bound K-BGT in a solid phase assay, i.e. peptide Na351-70 with a Kd-300 nM and peptide Ncy31-18 with slightly lower affinity (Kd-500 uM). Preincubation of 12?-~-BGT with peptides Na351-70 or Na31-18 resulted in >90% inhibition of K-'~'I-BGT binding to native N-nAChR expressed on the neuronal cell line PC12. Under the same conditions, two additional peptides, Na3180-199 and Na3183-201, were found to inhibit K-12' I-BGT binding to PC12 by-50%. These latter peptides represent sequences that are homologous to those shown previously to bind a-bungarotoxin. Peptide Na351-70 (400 pM) also reduced by approximately 4-fold the observed rate of association of K-BGT to PC12 cells. The results of these experiments identify sequence segments of the lxg subunit which are likely to interact with K-BGT and may indicate the relative contribution that these segments make in the formation of the high affinity K-BGT-binding site of this N-nAChR subtype. Neuronal nicotinic cholinergic receptors (N-nAChRs)' are heteromeric proteins consisting of at least two types of subunits, (Y and p (l-3). Sequences for three different but homologous (Y subunits (CQ, CQ, and a4) and two fl subunits (pZ and p3) have been reported for the peripheral and central nervous systems of the rat (1, 4-7). One unique structural feature of all (Y subunits is the presence of 2 adjacent cysteine residues contained in a putative extracellular domain between

Research paper thumbnail of Identification of a brain acetylcholine receptor alpha subunit able to bind alpha-bungarotoxin

Journal of Biological Chemistry, 1990

Peptides corresponding to sequence segments homologous to an alpha-bungarotoxin (alpha-BGT) bindi... more Peptides corresponding to sequence segments homologous to an alpha-bungarotoxin (alpha-BGT) binding region on the alpha subunit of the Torpedo nicotinic cholinergic receptor (nAChR) were synthesized for each identified nAChR alpha subunit of the rat nervous system (alpha 1, which is expressed in muscle, and alpha 2, alpha 3, alpha 4, and alpha 5, which are expressed by neurons). The peptides were tested for their ability to directly bind 125I-alpha-BGT and to compete for 125I-alpha-BGT with Torpedo nAChR and with the alpha-BGT-binding component expressed by PC12, a sympathetic neuronal cell line. In addition to peptides of the muscle alpha 1 subunit, peptides corresponding to the sequence of a neuronal subunit, alpha 5, were able to bind 125I-alpha-BGT. Peptides containing the sequence segments 182-201 of the alpha 1 subunit and 180-199 of the alpha 5 subunit competed with Torpedo nAChR for 125I-alpha-BGT binding with IC50 values of 0.5 and 3.5 microM, respectively. Both of these peptides were also able to compete for 125I-alpha-BGT binding with native Torpedo nAChR and with the alpha-BGT-binding protein(s) expressed on PC12 cells. To determine if other sequence segments contribute to form the neuronal alpha-BGT-binding site, overlapping peptides corresponding to the putative extracellular domain of the alpha 5 subunit were synthesized and used both in direct binding assays and in competition experiments. Peptides corresponding to amino acids 16-35 and 180-199 of the alpha 5 subunit directly bound 125I-alpha-BGT and inhibited the binding of toxin to both Torpedo nAChR and PC12 cells. The results of these studies strongly support identification of the alpha 5 subunit as a component of a neuronal alpha-BGT-binding nAChR.

Research paper thumbnail of Monoclonal antibodies FK1 and WF6 define two neighboring ligand binding sites on Torpedo acetylcholine receptor alpha-polypeptide

Journal of Biological Chemistry, 1994

Previous studies have identified the sequence region flanking the invariant vicinal cysteinyl res... more Previous studies have identified the sequence region flanking the invariant vicinal cysteinyl residues at positions 192 and 193 of the nicotinic acetylcholine receptor alpha-subunit as containing major elements of the binding site for acetylcholine and its agonists and antagonists, including antibody WF6 (Conti-Tronconi, B. M., Diethelm, B. M., Wu, X., Tang, F., Bertazzon, T., Schröder, B., Reinhardt-Maelicke, A., and Maelicke, A. (1991) Biochemistry 30, 2575-2584). Recently we have shown that the sequence region flanking lysine alpha 125 contains elements of the binding site for physostigmine and related ligands, including antibody FK1 (Schrattenholz, A., Godovac-Zimmerman, J., Schäfer, H.-J., Albuquerque, E. X., and Maelicke, A. (1993) Eur. J. Biochem. 216, 671-677). Here we report the identification by enzyme-linked immunosorbent assay techniques, employing fragments of the Torpedo nicotinic acetylcholine receptor alpha-subunit N-terminal region and a panel of synthetic peptides matching in sequence preselected portions of this subunit, of the sequence regions alpha 118-145 and alpha 181-216 as contributing to the FK1 epitope. Of the synthetic peptides employed, alpha 118-137 displayed the highest affinity of FK1 binding. Binding of FK1 and WF6 to single residue-substituted analogs of the sequence alpha 181-200 indicated that the two antibodies have different attachment point patterns within this sequence region. These results, and those of ligand competition studies, suggest that the binding sites for FK1 and physostigmine, and those of WF6 and acetylcholine, are within the same general region of the receptor's three-dimensional structure. The sites neighbor each other, with limited overlap in the case of occupation by high molecular weight ligands.

Research paper thumbnail of Antibodies as Tools to Study the Structure of Membrane Proteins: The Case of the Nicotine Acetylcholine Receptor

Annual Review of Biophysics and Biomolecular Structure, 1996

The nicotinic acetylcholine receptor is the prototype of the ionotropic receptor superfamily of p... more The nicotinic acetylcholine receptor is the prototype of the ionotropic receptor superfamily of proteins, which includes the closely related gamma- aminobutyric acid type A and glycine receptors, and more distantly related serotonin type-3 and glutamate receptors. Several models of the transmembrane topology of the nicotinic acetylcholine receptor subunits were originally proposed based on hydropathy analysis of their deduced amino acid sequences. Antibodies specific to different epitopes of the nicotinic acetylcholine receptor have proven to be valuable probes for examining the validity of those models. Despite important caveats, a viable model for the transmembrane structure and functional topology of the nicotinic acetylcholine receptor subunits has been obtained from the antibody mapping studies. This model, and the associated methodological shortcomings and obstacles that were overcome in the process of its formulation, can legitimately be extended to other members of the ionotropic receptor superfamily and to other membrane proteins as well.

Research paper thumbnail of Anti-TNFα antibodies and methods of use

Research paper thumbnail of Anti-TNFalpha antibodies and methods of use

Research paper thumbnail of The Nicotinic Acetylcholine Receptor as a Model for a Superfamily of Ligand-Gated Ion Channel Proteins

Protein Engineering and Design, 1996

Research paper thumbnail of Use of Synthetic Peptides and High Affinity Protein Ligands for Structural Studies of Central and Peripheral Nicotinic Receptors

Molecular Biology of Neuroreceptors and Ion Channels, 1989

Neurotransmitter receptors are key components of neuronal function. A major breakthrough in their... more Neurotransmitter receptors are key components of neuronal function. A major breakthrough in their study has been provided by cloning and sequencing of their genes and deduction of the amino acid sequence of their precursors.

Research paper thumbnail of A definitive source?

Trends in Biotechnology, 2000

Research paper thumbnail of Binding of native κ-neurotoxins and site-directed mutants to nicotinic acetylcholine receptors

Research paper thumbnail of Transplantation of a 17-amino acid alpha-helical DNA-binding domain into an antibody molecule confers sequence-dependent DNA recognition

Proceedings of the National Academy of Sciences, 1995

Recombinant antibodies capable of sequence-specific interactions with nucleic acids represent a c... more Recombinant antibodies capable of sequence-specific interactions with nucleic acids represent a class of DNA- and RNA-binding proteins with potential for broad application in basic research and medicine. We describe the rational design of a DNA-binding antibody, Fab-Ebox, by replacing a variable segment of the immunoglobulin heavy chain with a 17-amino acid domain derived from TFEB, a class B basic helix-loop-helix protein. DNA-binding activity was studied by electrophoretic mobility-shift assays in which Fab-Ebox was shown to form a specific complex with DNA containing the TFEB recognition motif (CACGTG). Similarities were found in the abilities of TFEB and Fab-Ebox to discriminate between oligodeoxyribonucleotides containing altered recognition sequences. Comparable interference of binding by methylation of cytosine residues indicated that Fab-Ebox and TFEB both contact DNA through interactions along the major groove of double-stranded DNA. The results of this study indicate that ...

Research paper thumbnail of Use of Synthetic Sequences of the Nicotinic Acetylcholine Receptor to Identify Structural Determinants of Binding Sites for Neurotoxins and Antibodies to the Main Immunogenic Region

Methods, 1993

Abstract Synthetic peptide sequences of the nicotinic acetylcholine receptor (AChR) have been use... more Abstract Synthetic peptide sequences of the nicotinic acetylcholine receptor (AChR) have been used extensively to identify structural features of two functionally important domains of the AChR: the binding site for cholinergic ligands and the main immunogenic region (MIR) (i.e., the set of overlapping epitopes that dominates the antibody response in the autoimmune disease myasthenia gravis) studies that used synthetic peptides have identified sequence regions and individual residues likely to contribute to formation of those domains. The results of several synthetic peptide investigations have been verified by different approaches, such as affinity labeling and study of AChRs carrying mutations of particular residues. Therefore, at least for certain protein ligands, the use of synthetic peptide sequences as representative structural elements of the corresponding receptor has demonstrated a reliable predictive value. Synthetic peptide studies enable, in a relatively short time, identification of candidate sequence regions for contributing to the structure of binding domains involved in protein/protein interaction. They serve as a foundation to direct the focus of more labor-intensive studies involving receptors mutated at individual residues to investigate their binding properties. We describe here the different types of assays employed in our laboratory using synthetic peptide strategies to investigate the AChR sequence regions contributing to the MIR and to cholinergic binding sites. The results obtained are summarized and critically compared and contrasted with those of other approaches. The reliability of the conclusions drawn from the aggregated results of those studies is discussed.

Research paper thumbnail of Transplantation of a 17-Amino Acid -Helical DNA-Binding Domain Into an Antibody Molecule Confers Sequence-Dependent DNA Recognition

Research paper thumbnail of The Nicotinic Acetylcholine Receptor: Structure and Autoimmune Pathology

Critical Reviews in Biochemistry and Molecular Biology, 1994

ABSTRACT

Research paper thumbnail of anti-TNF antibodies and methods of use

An antibody that can bind TNF alpha, wherein the antibody comprises: (a) a polypeptide comprising... more An antibody that can bind TNF alpha, wherein the antibody comprises: (a) a polypeptide comprising a polypeptide having the sequence shown in SEQ ID NO: 2; and (b) a polypeptide comprising a polypeptide having the sequence shown in SEQ ID NO: 380, wherein said antibody binds to TNF alpha higher than infliximab affinity as determined using a Biacore assay and in the the VH region infliximab has the sequence shown in SEQ ID NO: 1 and the VL of infliximab has the sequence shown in SEQ ID NO: 379.

Research paper thumbnail of Polypeptides de liaison de chaine variable de type immunoglobuline et procedes d'utilisation

Cette invention concerne une population variee isolee de polypeptides de liaison de type VH. Chaq... more Cette invention concerne une population variee isolee de polypeptides de liaison de type VH. Chaque polypeptide de liaison de cette population comprend une combinaison non certifiee d'un polypeptide code par exon de zone VH d'immunoglobuline, d'un polypeptide code par exon de zone JH et d'un polypeptide code par exon de zone D, ces polypeptides codes par exon de zone VH, D et JH sont joints en un seul polypeptide formant un polypeptide de liaison de type immunoglobuline VH, ou un fragment fonctionnel de celui-ci.

Research paper thumbnail of Species- and subtype-specitid recognition by antibody WF6 of a sequence segment forming an α-bungaratoxin binding site on the nictoinic acetylcholine receptor α subunit

Journal of Receptor Research, 1992

Research paper thumbnail of Immunoglobulin-like variable chain binding polypeptides and methods of use

Research paper thumbnail of Anti-TNFα antibodies

Research paper thumbnail of Identification of sequence segments forming the alpha-bungarotoxin binding sites on two nicotinic acetylcholine receptor alpha subunits from the avian brain

Journal of Biological Chemistry, 1991

Research paper thumbnail of Localization of sequence segments forming a kappa-bungarotoxin-binding site on the alpha 3 neuronal nicotinic receptor

Journal of Biological Chemistry, 1990

To identify the sequence segments of the a3 subunit of the neuronal nicotinic acetylcholine recep... more To identify the sequence segments of the a3 subunit of the neuronal nicotinic acetylcholine receptor (N-nAChR) forming the binding site for the cholinergic antagonist K-bungarotoxin (K-BGT), overlapping peptides corresponding to the complete a3 sequence were tested for their ability to bind 12'1-labeled K-BGT. Two peptides located within the N-terminal extracellular domain specifically bound K-BGT in a solid phase assay, i.e. peptide Na351-70 with a Kd-300 nM and peptide Ncy31-18 with slightly lower affinity (Kd-500 uM). Preincubation of 12?-~-BGT with peptides Na351-70 or Na31-18 resulted in >90% inhibition of K-'~'I-BGT binding to native N-nAChR expressed on the neuronal cell line PC12. Under the same conditions, two additional peptides, Na3180-199 and Na3183-201, were found to inhibit K-12' I-BGT binding to PC12 by-50%. These latter peptides represent sequences that are homologous to those shown previously to bind a-bungarotoxin. Peptide Na351-70 (400 pM) also reduced by approximately 4-fold the observed rate of association of K-BGT to PC12 cells. The results of these experiments identify sequence segments of the lxg subunit which are likely to interact with K-BGT and may indicate the relative contribution that these segments make in the formation of the high affinity K-BGT-binding site of this N-nAChR subtype. Neuronal nicotinic cholinergic receptors (N-nAChRs)' are heteromeric proteins consisting of at least two types of subunits, (Y and p (l-3). Sequences for three different but homologous (Y subunits (CQ, CQ, and a4) and two fl subunits (pZ and p3) have been reported for the peripheral and central nervous systems of the rat (1, 4-7). One unique structural feature of all (Y subunits is the presence of 2 adjacent cysteine residues contained in a putative extracellular domain between

Research paper thumbnail of Identification of a brain acetylcholine receptor alpha subunit able to bind alpha-bungarotoxin

Journal of Biological Chemistry, 1990

Peptides corresponding to sequence segments homologous to an alpha-bungarotoxin (alpha-BGT) bindi... more Peptides corresponding to sequence segments homologous to an alpha-bungarotoxin (alpha-BGT) binding region on the alpha subunit of the Torpedo nicotinic cholinergic receptor (nAChR) were synthesized for each identified nAChR alpha subunit of the rat nervous system (alpha 1, which is expressed in muscle, and alpha 2, alpha 3, alpha 4, and alpha 5, which are expressed by neurons). The peptides were tested for their ability to directly bind 125I-alpha-BGT and to compete for 125I-alpha-BGT with Torpedo nAChR and with the alpha-BGT-binding component expressed by PC12, a sympathetic neuronal cell line. In addition to peptides of the muscle alpha 1 subunit, peptides corresponding to the sequence of a neuronal subunit, alpha 5, were able to bind 125I-alpha-BGT. Peptides containing the sequence segments 182-201 of the alpha 1 subunit and 180-199 of the alpha 5 subunit competed with Torpedo nAChR for 125I-alpha-BGT binding with IC50 values of 0.5 and 3.5 microM, respectively. Both of these peptides were also able to compete for 125I-alpha-BGT binding with native Torpedo nAChR and with the alpha-BGT-binding protein(s) expressed on PC12 cells. To determine if other sequence segments contribute to form the neuronal alpha-BGT-binding site, overlapping peptides corresponding to the putative extracellular domain of the alpha 5 subunit were synthesized and used both in direct binding assays and in competition experiments. Peptides corresponding to amino acids 16-35 and 180-199 of the alpha 5 subunit directly bound 125I-alpha-BGT and inhibited the binding of toxin to both Torpedo nAChR and PC12 cells. The results of these studies strongly support identification of the alpha 5 subunit as a component of a neuronal alpha-BGT-binding nAChR.

Research paper thumbnail of Monoclonal antibodies FK1 and WF6 define two neighboring ligand binding sites on Torpedo acetylcholine receptor alpha-polypeptide

Journal of Biological Chemistry, 1994

Previous studies have identified the sequence region flanking the invariant vicinal cysteinyl res... more Previous studies have identified the sequence region flanking the invariant vicinal cysteinyl residues at positions 192 and 193 of the nicotinic acetylcholine receptor alpha-subunit as containing major elements of the binding site for acetylcholine and its agonists and antagonists, including antibody WF6 (Conti-Tronconi, B. M., Diethelm, B. M., Wu, X., Tang, F., Bertazzon, T., Schröder, B., Reinhardt-Maelicke, A., and Maelicke, A. (1991) Biochemistry 30, 2575-2584). Recently we have shown that the sequence region flanking lysine alpha 125 contains elements of the binding site for physostigmine and related ligands, including antibody FK1 (Schrattenholz, A., Godovac-Zimmerman, J., Schäfer, H.-J., Albuquerque, E. X., and Maelicke, A. (1993) Eur. J. Biochem. 216, 671-677). Here we report the identification by enzyme-linked immunosorbent assay techniques, employing fragments of the Torpedo nicotinic acetylcholine receptor alpha-subunit N-terminal region and a panel of synthetic peptides matching in sequence preselected portions of this subunit, of the sequence regions alpha 118-145 and alpha 181-216 as contributing to the FK1 epitope. Of the synthetic peptides employed, alpha 118-137 displayed the highest affinity of FK1 binding. Binding of FK1 and WF6 to single residue-substituted analogs of the sequence alpha 181-200 indicated that the two antibodies have different attachment point patterns within this sequence region. These results, and those of ligand competition studies, suggest that the binding sites for FK1 and physostigmine, and those of WF6 and acetylcholine, are within the same general region of the receptor's three-dimensional structure. The sites neighbor each other, with limited overlap in the case of occupation by high molecular weight ligands.

Research paper thumbnail of Antibodies as Tools to Study the Structure of Membrane Proteins: The Case of the Nicotine Acetylcholine Receptor

Annual Review of Biophysics and Biomolecular Structure, 1996

The nicotinic acetylcholine receptor is the prototype of the ionotropic receptor superfamily of p... more The nicotinic acetylcholine receptor is the prototype of the ionotropic receptor superfamily of proteins, which includes the closely related gamma- aminobutyric acid type A and glycine receptors, and more distantly related serotonin type-3 and glutamate receptors. Several models of the transmembrane topology of the nicotinic acetylcholine receptor subunits were originally proposed based on hydropathy analysis of their deduced amino acid sequences. Antibodies specific to different epitopes of the nicotinic acetylcholine receptor have proven to be valuable probes for examining the validity of those models. Despite important caveats, a viable model for the transmembrane structure and functional topology of the nicotinic acetylcholine receptor subunits has been obtained from the antibody mapping studies. This model, and the associated methodological shortcomings and obstacles that were overcome in the process of its formulation, can legitimately be extended to other members of the ionotropic receptor superfamily and to other membrane proteins as well.

Research paper thumbnail of Anti-TNFα antibodies and methods of use

Research paper thumbnail of Anti-TNFalpha antibodies and methods of use

Research paper thumbnail of The Nicotinic Acetylcholine Receptor as a Model for a Superfamily of Ligand-Gated Ion Channel Proteins

Protein Engineering and Design, 1996

Research paper thumbnail of Use of Synthetic Peptides and High Affinity Protein Ligands for Structural Studies of Central and Peripheral Nicotinic Receptors

Molecular Biology of Neuroreceptors and Ion Channels, 1989

Neurotransmitter receptors are key components of neuronal function. A major breakthrough in their... more Neurotransmitter receptors are key components of neuronal function. A major breakthrough in their study has been provided by cloning and sequencing of their genes and deduction of the amino acid sequence of their precursors.

Research paper thumbnail of A definitive source?

Trends in Biotechnology, 2000

Research paper thumbnail of Binding of native κ-neurotoxins and site-directed mutants to nicotinic acetylcholine receptors

Research paper thumbnail of Transplantation of a 17-amino acid alpha-helical DNA-binding domain into an antibody molecule confers sequence-dependent DNA recognition

Proceedings of the National Academy of Sciences, 1995

Recombinant antibodies capable of sequence-specific interactions with nucleic acids represent a c... more Recombinant antibodies capable of sequence-specific interactions with nucleic acids represent a class of DNA- and RNA-binding proteins with potential for broad application in basic research and medicine. We describe the rational design of a DNA-binding antibody, Fab-Ebox, by replacing a variable segment of the immunoglobulin heavy chain with a 17-amino acid domain derived from TFEB, a class B basic helix-loop-helix protein. DNA-binding activity was studied by electrophoretic mobility-shift assays in which Fab-Ebox was shown to form a specific complex with DNA containing the TFEB recognition motif (CACGTG). Similarities were found in the abilities of TFEB and Fab-Ebox to discriminate between oligodeoxyribonucleotides containing altered recognition sequences. Comparable interference of binding by methylation of cytosine residues indicated that Fab-Ebox and TFEB both contact DNA through interactions along the major groove of double-stranded DNA. The results of this study indicate that ...

Research paper thumbnail of Use of Synthetic Sequences of the Nicotinic Acetylcholine Receptor to Identify Structural Determinants of Binding Sites for Neurotoxins and Antibodies to the Main Immunogenic Region

Methods, 1993

Abstract Synthetic peptide sequences of the nicotinic acetylcholine receptor (AChR) have been use... more Abstract Synthetic peptide sequences of the nicotinic acetylcholine receptor (AChR) have been used extensively to identify structural features of two functionally important domains of the AChR: the binding site for cholinergic ligands and the main immunogenic region (MIR) (i.e., the set of overlapping epitopes that dominates the antibody response in the autoimmune disease myasthenia gravis) studies that used synthetic peptides have identified sequence regions and individual residues likely to contribute to formation of those domains. The results of several synthetic peptide investigations have been verified by different approaches, such as affinity labeling and study of AChRs carrying mutations of particular residues. Therefore, at least for certain protein ligands, the use of synthetic peptide sequences as representative structural elements of the corresponding receptor has demonstrated a reliable predictive value. Synthetic peptide studies enable, in a relatively short time, identification of candidate sequence regions for contributing to the structure of binding domains involved in protein/protein interaction. They serve as a foundation to direct the focus of more labor-intensive studies involving receptors mutated at individual residues to investigate their binding properties. We describe here the different types of assays employed in our laboratory using synthetic peptide strategies to investigate the AChR sequence regions contributing to the MIR and to cholinergic binding sites. The results obtained are summarized and critically compared and contrasted with those of other approaches. The reliability of the conclusions drawn from the aggregated results of those studies is discussed.