Positive charges in a putative amphiphilic helix in the carboxyl-terminal region of the third intracellular loop of the luteinizing hormone/chorionic gonadotropin receptor are not required for hormone-stimulated cAMP production but are necessary for expression of the receptor at the plasma membrane.
The LH/CG receptor (LHR) is a member of the family of G protein-coupled receptors and activates Gs when stimulated by LH or CG. Studies from other G protein-coupled receptors have implicated the carboxyl-terminal region of the third intracellular loop as being involved in the activation of G proteins. It has been suggested that the potential ability of this region to form an amphiphilic helix, with positively charged residues aligned to one face, may be important for this biological activity. To test whether the positively charged lysine residues, and thus an amphiphilic helix, in the carboxyl terminal region of the rat LHR (rLHR) are indeed important in the activation of Gs by the rLHR, a mutant rLHR was constructed in which lysines 541, 544, and 557 were simultaneously substituted with alanines. Clonal 293 cells expressing comparable numbers of cell-surface recombinant wild type rLHR or rLHR(K541,544,547A) were generated. Cells expressing the mutant receptor-bound human CG (hCG) with the same high affinity as those expressing the wild type rLHR. Since the numbers of receptors and binding affinities between the two cell lines were comparable, any changes in basal or hCG stimulated cAMP production could readily be interpreted as an alteration in the mutant receptor's ability to activate Gs. It was found, however, that basal cAMP production, the concentration of hCG required to elicit half-maximal cAMP production, and the maximal levels of cAMP produced in response to hCG were all unchanged in cells expressing rLHR(K541,544,547A).(ABSTRACT TRUNCATED AT 250 WORDS)
Wang
H
H
Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City 52242.
Jaquette
J
J
Collison
K
K
Segaloff
D L
DL
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D006063:Chorionic Gonadotropin / Q000502:physiology*; D000242:Cyclic AMP / Q000096:biosynthesis*; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011974:Receptors, LH / Q000737:chemistry* / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship
D006063:Chorionic Gonadotropin; D011974:Receptors, LH; D011993:Recombinant Fusion Proteins; D000242:Cyclic AMP; D019204:GTP-Binding Proteins