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Cysteine scanning mutagenesis of transmembrane domain V of the M1 muscarinic receptor
Vol. 60, Nos. 13114, 1997
1177
Abstracts
29 CYSTEINE SCANNING THE Ml MUSCARINIC
MUTAGENESIS RECEPTOR.
OF TRANSMEMBRANE
K. Allman, K.M. Page, and E.C. Hulme, MRC National Mill Hill, London NW7 lAA, U.K.
Institute
DOMAIN
V OF
for Medical Research,
The aim of this study was to make positive identifications of amino acids in transmembrane domain (TMD) V of the ml muscarinic receptor (mAChR) which contact the acetylcholine sidechain. The principal approach used was cysteine-substitution mutagenesis followed by chemical reaction. Consecutive residues (Ile188-Ala196) in TMD V of the rat ml mAChR were mutated to cysteine, and to alanine or glycine. Mutant receptors were probed by binding of the antagonist [3H]N-methylscopolamine ([3H]NMS), the agonist acetylcholine, and the sulfhydryl-alkylating ACh analogue, bromoacetylcholine (BrACh). Only the mutation of Ala193 affected the binding of r3H]NMS, causing a 5-fold decrease in affinity. In contrast, ACh affinity was strongly decreased by the mutation of Ile188, Thr189, Thr192, and Ala196, residues which may be modelled as lying on one face of an a-helix. Interestingly, Phel9OCys caused a significant increase in ACh affinity. The reversible binding of BrACh resembled that of ACh, and was similarly affected by the mutations. In addition, BrACh caused rapid, irreversible blockade of the Thrl92Cys mutant, and showed evidence of a lower rate of reaction with Alal93Cys, Ilel88Cys, and Alal96Cys, but not with the other Cys mutants, or the wild-type receptor. These observations pinpoint Thr192 as a primary candidate for contact with the acetyl methyl group of ACh, and suggest that a penumbra of surrounding residues, particularly Ala196, may also participate in a putative methyl binding pocket.
30 PHENOTYPIC INTERCONVERSION OF MUSCARINIC RECEPTOR SUBTYPES COUPLED TO PHOSPHOLIPASE C BY EXCHANGING TWO AMINO ACIDS. N.S.M. Geoghagen and N.H. Lee. Department of Molecular and Cellular Biology. for Genomic Research, Rockville, MD 20850, USA.
The Institute
Comparison of the ml, m3 and m5 muscarinic acetylcholine receptor (mAChR) sequences reveals that the terminal segments of the third intracellular (i-3) loop are well conserved with the exception of two amino acids. The amino- and carboxyl-terminal segments of the ml mAChR correspond to the sequences RIYRETENR and LVKEKKAAE (underlined amino acids in the ml sequence are not conserved in the m3 and m5 sequences), respectively. The KKAAR motif, contained within the LVKEKKAAR segment of the ml mAChR, is critical for agonist-mediated signaling (Lee et al., Mol. Pharmacol. 50: 140-148, 1996). In both the m3 and m5 mAChRs, the amino- and carboxyl-terminal sequences are RIY(IVR)ETEKR and L(YV)KEKKAAQ, respectively. Carbachol (CBC) increases phosphoinositide (PI) hydrolysis in Chinese hamster ovary (CHO) cells transfected with the ml, m3 or m5 mAChR subtype. The magnitude of this increase is density- and subtype-dependent. Titration of wild-type m3 mAChR densities in CHO cells to -100 fmol/mg protein yielded no PI response following CBC challenge. In cells expressing wild-type ml mAChRs at similar densities, CBC elicited a 4-fold increase. To determine if the two non-conserved amino acids in the membrane proximal i-3 loop are responsible for these signaling differences, we have mutated these residues and expressed the mutant receptors in CHO cells. Mutation of the non-conserved amino acids of the m3 mAChR to the corresponding residues in the ml sequence yielded an m3 mutant with a PI response that phenotypically resembles the wild-type ml mAChR. Conversely, a wild-type m3 phenotype was displayed when either one of these non-conserved residues in the ml mAChR was mutated into the corresponding residue found in the m3 sequence.
1177
Abstracts
29 CYSTEINE SCANNING THE Ml MUSCARINIC
MUTAGENESIS RECEPTOR.
OF TRANSMEMBRANE
K. Allman, K.M. Page, and E.C. Hulme, MRC National Mill Hill, London NW7 lAA, U.K.
Institute
DOMAIN
V OF
for Medical Research,
The aim of this study was to make positive identifications of amino acids in transmembrane domain (TMD) V of the ml muscarinic receptor (mAChR) which contact the acetylcholine sidechain. The principal approach used was cysteine-substitution mutagenesis followed by chemical reaction. Consecutive residues (Ile188-Ala196) in TMD V of the rat ml mAChR were mutated to cysteine, and to alanine or glycine. Mutant receptors were probed by binding of the antagonist [3H]N-methylscopolamine ([3H]NMS), the agonist acetylcholine, and the sulfhydryl-alkylating ACh analogue, bromoacetylcholine (BrACh). Only the mutation of Ala193 affected the binding of r3H]NMS, causing a 5-fold decrease in affinity. In contrast, ACh affinity was strongly decreased by the mutation of Ile188, Thr189, Thr192, and Ala196, residues which may be modelled as lying on one face of an a-helix. Interestingly, Phel9OCys caused a significant increase in ACh affinity. The reversible binding of BrACh resembled that of ACh, and was similarly affected by the mutations. In addition, BrACh caused rapid, irreversible blockade of the Thrl92Cys mutant, and showed evidence of a lower rate of reaction with Alal93Cys, Ilel88Cys, and Alal96Cys, but not with the other Cys mutants, or the wild-type receptor. These observations pinpoint Thr192 as a primary candidate for contact with the acetyl methyl group of ACh, and suggest that a penumbra of surrounding residues, particularly Ala196, may also participate in a putative methyl binding pocket.
30 PHENOTYPIC INTERCONVERSION OF MUSCARINIC RECEPTOR SUBTYPES COUPLED TO PHOSPHOLIPASE C BY EXCHANGING TWO AMINO ACIDS. N.S.M. Geoghagen and N.H. Lee. Department of Molecular and Cellular Biology. for Genomic Research, Rockville, MD 20850, USA.
The Institute
Comparison of the ml, m3 and m5 muscarinic acetylcholine receptor (mAChR) sequences reveals that the terminal segments of the third intracellular (i-3) loop are well conserved with the exception of two amino acids. The amino- and carboxyl-terminal segments of the ml mAChR correspond to the sequences RIYRETENR and LVKEKKAAE (underlined amino acids in the ml sequence are not conserved in the m3 and m5 sequences), respectively. The KKAAR motif, contained within the LVKEKKAAR segment of the ml mAChR, is critical for agonist-mediated signaling (Lee et al., Mol. Pharmacol. 50: 140-148, 1996). In both the m3 and m5 mAChRs, the amino- and carboxyl-terminal sequences are RIY(IVR)ETEKR and L(YV)KEKKAAQ, respectively. Carbachol (CBC) increases phosphoinositide (PI) hydrolysis in Chinese hamster ovary (CHO) cells transfected with the ml, m3 or m5 mAChR subtype. The magnitude of this increase is density- and subtype-dependent. Titration of wild-type m3 mAChR densities in CHO cells to -100 fmol/mg protein yielded no PI response following CBC challenge. In cells expressing wild-type ml mAChRs at similar densities, CBC elicited a 4-fold increase. To determine if the two non-conserved amino acids in the membrane proximal i-3 loop are responsible for these signaling differences, we have mutated these residues and expressed the mutant receptors in CHO cells. Mutation of the non-conserved amino acids of the m3 mAChR to the corresponding residues in the ml sequence yielded an m3 mutant with a PI response that phenotypically resembles the wild-type ml mAChR. Conversely, a wild-type m3 phenotype was displayed when either one of these non-conserved residues in the ml mAChR was mutated into the corresponding residue found in the m3 sequence.