Random chimeragenesis of G-protein-coupled receptors. Mapping the affinity of the cAMP chemoattractant receptors in Dictyostelium

J. Y. Kim, P. N. Devreotes

Research output: Contribution to journalArticlepeer-review

Abstract

G-protein-coupled receptors mediate a wide variety of responses to extracellular stimuli in eucaryotic cells. Binding of the ligand to these receptors is thought to involve contacts within a pocket formed by the seven transmembrane domains inferred from the sequences of these genes. A family of four surface cAMP receptors that mediate responses to secreted cAMP coordinates the developmental program of Dictyostelium. A large difference in affinity for cAMP exists between cAR1 (25 and 230 nM) and cAR2 (>5 μM). To understand the basis for this affinity difference, we generated an extensive series of cAR1/cAR2 and cAR2/cAR1 chimeras using a technique designated 'random chimeragenesis.' When a linearized plasmid was transformed into Escherichia coli, tandemly positioned cAR1 and cAR2 genes crossed over at homologous regions. The cAMP binding properties and EC50 values for agonist-induced phosphorylation of each of the chimeras were characterized in order to map the domains that determine the affinity. These studies implicated a domain in the second extracellular loop in which only 5 residues differ between the two receptors as the major determinant of affinity. A secondary domain including residues 110-147 (11 residue differences) was identified as a minor determinant of affinity.

Original languageEnglish (US)
Pages (from-to)28724-28731
Number of pages8
JournalJournal of Biological Chemistry
Volume269
Issue number46
StatePublished - 1994

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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