A dileucine-based motif in the C-terminal tail of the lutropin/choriogonadotropin receptor inhibits endocytosis of the agonist-receptor complex.
The rat lutropin/choriogonadotropin receptor (rLHR) is a member of the rhodopsin-like subfamily of G protein-coupled receptors that has two adjacent dileucine motifs in the C-terminal cytoplasmic tail. Here we show that simultaneous (L613,614,615,616A) or individual (L613,L614A or L615,616A) mutation of the two adjacent dileucine motifs to alanines results in mutants with enhanced rates of agonist-induced internalization. The L613,L614A mutation was much more effective in enhancing internalization than the L615,L616A mutation. Moreover, the L613A mutation was more effective than the L614A mutation. Because in the human LHR the residues equivalent to L613 and L614 of the rLHR are a phenylalanine and a leucine (F635 and L636), we also prepared mutants that exchanged these motifs. In the rLHR, an LL-to-FL exchange enhanced endocytosis, and in the human LHR, an FL-to-LL exchange impaired endocytosis. The internalization of rLHR-wt and rLRH-L613,L614A was inhibited by coexpression of the clathrin-binding domain of beta-arrestin. In fact, this manipulation reduced the enhanced rate of internalization of rLHR-L613,614A back to that of rLHR-wt. The L613,614A mutation does not affect the degradation of the internalized agonist or the membrane targeting of the nascent rLHR. The L615,616A mutation also did not affect degradation of the internalized agonist but impaired the membrane targeting of the nascent rLHR. We conclude that the dileucine-based motifs of the rLHR inhibit internalization and suggest that this inhibition may be due to an impairment in the binding of the rLHR to endogenous nonvisual arrestins.
Nakamura
K
K
Department of Pharmacology, The University of Iowa, Iowa City, Iowa 52242-1109, USA.
Ascoli
M
M
D000595:Amino Acid Sequence; D000818:Animals; D019393:Arrestin / Q000378:metabolism; D001692:Biological Transport; D002478:Cells, Cultured; D006063:Chorionic Gonadotropin / Q000378:metabolism; D034281:Dynamins; D004705:Endocytosis; D020558:GTP Phosphohydrolases / Q000378:metabolism; D006801:Humans; D007930:Leucine / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D051381:Rats; D011974:Receptors, LH / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction
D019393:Arrestin; D006063:Chorionic Gonadotropin; D011974:Receptors, LH; D020558:GTP Phosphohydrolases; D034281:Dynamins; D007930:Leucine