Constitutive and agonist-dependent self-association of the cell surface human lutropin receptor.
The human lutropin receptor (hLHR) is a G protein-coupled receptor (GPCR) that plays an essential role in reproductive physiology. The present studies were undertaken to determine whether the hLHR self-associates. We show that high molecular weight complexes of the hLHR can be co-immunoprecipitated from 293 cells transfected with differentially tagged hLHRs. These complexes are detected only in extracts from cells that have been co-transfected and not in extracts combined from cells expressing only one form of tagged hLHR, confirming the in vivo self-association of the receptor. In transiently transfected cells, in which a small percentage of cells overexpress hLHR and most of the hLHR is located intracellularly in the ER, the self-associated hLHR is composed predominantly of immature hLHR. When cells were transiently co-transfected with wild-type hLHR and a misfolded mutant of the hLHR, a physical association of the ER-localized misfolded mutant with the immature hLHR was observed, resulting in a decreased cell surface expression of the wild-type receptor. In contrast, in stably transfected cells, where the majority of cells express receptor and there is much less intracellular accumulation of hLHR, the self-associated forms of the hLHR are composed predominantly of cell surface receptor. The abundance of cell surface hLHR dimers and oligomers, as detected on SDS gels, is increased further upon human choriogonadotropin treatment of the stably transfected cells. In addition to documenting the self-association of cell surface hLHR, our results underscore the importance of the cellular distribution of recombinant GPCR as it relates to the nature of the GPCR dimerization and oligomerization.
Department of Physiology and Biophysics, University of Iowa, Iowa City, Iowa 52242, USA.
Ya-Xiong
YX
Tao
Nathan B
NB
Johnson
Deborah L
DL
Segaloff
D019921:Biotinylation; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D000939:Epitopes; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000378:metabolism; D006062:Gonadotropins / Q000737:chemistry; D006801:Humans; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009154:Mutation; D011233:Precipitin Tests; D011485:Protein Binding; D017510:Protein Folding; D011974:Receptors, LH / Q000737:chemistry*; D014162:Transfection
D018076:DNA, Complementary; D003902:Detergents; D000939:Epitopes; D006062:Gonadotropins; D011974:Receptors, LH; D006026:Glycoside Hydrolases