Surface retention of an inactivating lutropin receptor mutant in exoloop 3.
The heptahelical lutropin receptor (LHR) signals primarily via the Gs-adenylyl cyclase pathway and undergoes ligand-mediated receptor desensitization and internalization. A loss-of-function rat LHR mutant was recently described in which a single amino acid residue replacement in exoloop 3, K583E, had no effect on human choriogonadotropin (hCG) binding but essentially abolished signaling. This LHR mutant is a prime candidate for which to study hCG-mediated receptor internalization since it is highly unlikely that an amino acid residue in exoloop 3 , i.e. an extracellular portion of LHR connecting transmembrane helices 6 and 7, could have any direct interaction with Galpha(s), which is located on the cytoplasmic face of the plasma membrane. A method to study endocytosis was adapted that involves concanavalin A binding to the glycoproteins on the cell surface, thus facilitating separation of the plasma membrane fraction from other cellular membrane fractions by sucrose gradient centrifugation. Conditions were used such that a single round of endocytosis could be determined with [125I]hCG. Endocytic rate constants of 0.03 and O min(-1) were obtained for LHR and the mutant, respectively, in transfected human embryonic kidney 293 cells; moreover, internalization of the mutant could not be restored by the addition of 8-Br-cAMP. Thus, the presence of the second messenger cAMP is not sufficient for internalization of ligand-occupied LHR. Rather, it appears that ligand-mediated activation and subsequent internalization of LHR results from an altered conformational state or a conformation-dependent post-ligand binding modification such as phosphorylation.
Bhowmick
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N
Department of Biochemistry and Molecular Biology, University of Georgia, Athens 30602, USA.
Narayan
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P
Puett
D
D
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002458:Cell Fractionation; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D006063:Chorionic Gonadotropin / Q000378:metabolism* / Q000494:pharmacology; D003208:Concanavalin A / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007668:Kidney; D011487:Protein Conformation; D051381:Rats; D011974:Receptors, LH / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D006063:Chorionic Gonadotropin; D006023:Glycoproteins; D011974:Receptors, LH; D011993:Recombinant Fusion Proteins; C026887:biotinylconcanavalin A; D003208:Concanavalin A; D015124:8-Bromo Cyclic Adenosine Monophosphate; D015544:Inositol 1,4,5-Trisphosphate; D000242:Cyclic AMP