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Characterization of a Region of the Lutropin Receptor Extracellular Domain Near Transmembrane Helix 1 That Is Important in Ligand-Mediated Signaling¹

Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602-7229

Address all correspondence and requests for reprints to: Dr. David Puett, Department of Biochemistry and Molecular Biology, Life Sciences Building, Green Street, University of Georgia, Athens, Georgia 30602-7229. E-mail: puett{at}bchiris.bmb.uga.edu

The lutropin receptor (LHR), a member of the G protein-coupled receptor family, contains a relatively large N-terminal extracellular domain, accounting for about half of the receptor and responsible for high affinity ligand binding, and a standard heptahelical portion with connecting loops and a C-terminal tail. LHR and the other two glycoprotein hormone receptors, i.e. the follitropin and TSH receptors, contain an invariant 10-amino acid residue sequence, FNPCEDIMGY (residues 328–337 in rat LHR), in the extracellular domain separated by only a few amino acid residues from the beginning of transmembrane helix 1. In view of the invariant nature of this region in the three glycoprotein hormone receptors and preliminary data in the literature on the importance of Glu³³² and Asp³³³ in signal transduction, we undertook a systematic investigation of all 10 amino acid residues because this region may function as a switch or trigger for communicating ligand binding to the extracellular domain with a conformational change of the membrane-embedded C-terminal half of the receptor to activate G proteins, particularly G_s. A total of 36 single, double, and multiple replacements, as well as two deletions, of LHR were prepared and characterized in transiently transfected COS-7 cells. Of these mutants LHRs, 26 expressed on the cell surface in sufficient numbers that quantitative assessments could be made of human choriogonadotropin binding and ligand-mediated cAMP production. Replacements of Cys³³¹ abolished ligand binding to intact cells, although binding could be detected after solubilization of the cells. Replacements of the other nine amino acid residues that did not interfere with receptor folding or trafficking had no significant effect on ligand binding affinity; however, replacements of Pro³³⁰, Glu³³², and Asp³³³ resulted in diminished signaling, especially for the two acidic residues. An interesting observation was made in which replacement of Tyr³³⁷ with Ala or Asp, while having no profound change on receptor function, could overcome to some extent limited expression of replacements at positions 332 and/or 333, thus permitting a more definitive analysis of signaling. Replacement of the decapeptide sequence with Gly₁₀ prevents expression, whereas deletion of all 10 residues and deletion of Glu³³²-Asp³³³ prevents functional expression at the cell surface. Thus, this invariant sequence in the glycoprotein hormones is required for proper folding, trafficking, and ligand-mediated signaling, but not ligand binding, in LHR. Amino acid residues, Glu³³², Asp³³³, and to a limited extent, Pro³³⁰, are important in ligand-mediated signaling but not ligand binding.

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