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Temperature Sensitivity of Some Mutants of the Lutropin/Choriogonadotropin Receptor¹

Department of Physiology and Biophysics, The University of Iowa College of Medicine, Iowa City, Iowa 52242

Address all correspondence and requests for reprints to: Deborah L. Segaloff, Ph.D., Department of Physiology and Biophysics, The University of Iowa College of Medicine, Iowa City, Iowa 52240.

The lutropin/choriogonadotropin receptor (LHR) is a G protein-coupled receptor central to reproductive physiology. In addition to the complex structure formed by seven membrane-spanning helices, this receptor also contains a large amino-terminal domain whose tertiary structure is unknown. Many mutations of this receptor result in partial or complete intracellular retention of the mutant, regardless of whether the mutations are located in the extracellular domain, interhelical loops, transmembrane helices, or cytoplasmic tail. Nonetheless, as long as a mutation has not disturbed a hormone binding site, the intracellularly trapped mutant retains high affinity binding for human CG (hCG), suggesting that it is not extremely misfolded. Because temperature is known to affect protein folding, we examined the effects of reduced temperature on cell surface expression of intracellularly retained mutants of the LHR. Our studies examined three different 293 cells lines, each stably expressing a different LHR mutant that is intracellularly retained at 37 C. The results presented demonstrate that preincubation of the cells for 48 h at 26 C markedly increased both the total hCG binding activity within each cell line as well as the percentage of binding activity at the cell surface. Furthermore, the cells bound hCG with a normal high affinity and responded to hCG with increased cAMP production normally. These data suggest that decreased temperatures can allow partially misfolded LHRs to fold properly and be expressed in functional form on the cell surface and thus present the potential for utilization of this approach for other intracellularly retained G protein-coupled receptor mutants.

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