Chimeric muscarinic cholinergic

β-adrenergic receptors that activate Gs in response to muscarinic agonists

Stephen K F Wong, Eric M. Parker, Elliott M. Ross

Research output: Contribution to journalArticle

171 Citations (Scopus)

Abstract

The M1-muscarinic cholinergic receptor (M1AChR) stimulates the release of inositol phosphates (IPs) but does not activate adenylyl cyclase. The β-adrenergic receptor (β-AR) stimulates adenylyl cyclase but has no effect on IP release. Amino acid sequences corresponding to the second (12) and third (13) intracellular loops of the turkey erythrocyte β-AR and a 12-amino acid segment near the N-terminal end of the 13 region were substituted into the corresponding regions of the human M1AChR. Chimeric receptors that contained either the entire 13 loop or the N-terminal dodecapeptide of that loop both mediated the 2-4-fold stimulation of adenylyl cyclase activity in membrane fractions of COS, A293, or Sf9 cells in response to carbachol. These chimeric receptors also retained the ability to stimulate IP release to the same extent as did the M1AChR. In COS cells transfected with the I3 chimeric receptor, the EC50 for carbachol was ∼7 μM for the stimulation of adenylyl cyclase and ∼2 μM for the release of IP; M1AChR-mediated IP release displayed an EC50 of ∼0.2 μM. Substitution of the I2 region of the β-AR into the M1AChR did not by itself alter selectivity for signaling. However, the I2+I3 and I2+dodecapeptide combined replacements stimulated adenylyl cyclase fully and caused at most 25% of the maximal stimulation of IP release observed with the M1AChR. Thus, a small region in the third cytoplasmic loop can alter the G proteins to which a receptor is coupled, but interaction among loops is evidently involved in fully determining G protein selectivity.

Original languageEnglish (US)
Pages (from-to)6219-6224
Number of pages6
JournalJournal of Biological Chemistry
Volume265
Issue number11
StatePublished - 1990

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Muscarinic Agonists
Inositol Phosphates
Cholinergic Receptors
Muscarinic Receptors
Adrenergic Receptors
Cholinergic Agents
Adenylyl Cyclases
COS Cells
Carbachol
GTP-Binding Proteins
Sf9 Cells
Amino Acids
Amino Acid Sequence
Substitution reactions
Erythrocytes
Membranes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Chimeric muscarinic cholinergic : β-adrenergic receptors that activate Gs in response to muscarinic agonists. / Wong, Stephen K F; Parker, Eric M.; Ross, Elliott M.

In: Journal of Biological Chemistry, Vol. 265, No. 11, 1990, p. 6219-6224.

Research output: Contribution to journalArticle

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abstract = "The M1-muscarinic cholinergic receptor (M1AChR) stimulates the release of inositol phosphates (IPs) but does not activate adenylyl cyclase. The β-adrenergic receptor (β-AR) stimulates adenylyl cyclase but has no effect on IP release. Amino acid sequences corresponding to the second (12) and third (13) intracellular loops of the turkey erythrocyte β-AR and a 12-amino acid segment near the N-terminal end of the 13 region were substituted into the corresponding regions of the human M1AChR. Chimeric receptors that contained either the entire 13 loop or the N-terminal dodecapeptide of that loop both mediated the 2-4-fold stimulation of adenylyl cyclase activity in membrane fractions of COS, A293, or Sf9 cells in response to carbachol. These chimeric receptors also retained the ability to stimulate IP release to the same extent as did the M1AChR. In COS cells transfected with the I3 chimeric receptor, the EC50 for carbachol was ∼7 μM for the stimulation of adenylyl cyclase and ∼2 μM for the release of IP; M1AChR-mediated IP release displayed an EC50 of ∼0.2 μM. Substitution of the I2 region of the β-AR into the M1AChR did not by itself alter selectivity for signaling. However, the I2+I3 and I2+dodecapeptide combined replacements stimulated adenylyl cyclase fully and caused at most 25{\%} of the maximal stimulation of IP release observed with the M1AChR. Thus, a small region in the third cytoplasmic loop can alter the G proteins to which a receptor is coupled, but interaction among loops is evidently involved in fully determining G protein selectivity.",
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