Aspartic Acid 564 in the Third Cytoplasmic Loop of the Luteinizing Hormone/Choriogonadotropin Receptor Is Crucial for Phosphorylation-independent Interaction with Arrestin2
pmid: 11867621
Aspartic Acid 564 in the Third Cytoplasmic Loop of the Luteinizing Hormone/Choriogonadotropin Receptor Is Crucial for Phosphorylation-independent Interaction with Arrestin2
Arrestin2 binding to the active but unphosphorylated luteinizing hormone/choriogonadotropin receptor (LH/CG R) in ovarian follicles is triggered by activation of ADP-ribosylation factor 6 (ARF6) and leads to uncoupling of this receptor from cAMP signaling. We sought to determine how arrestin2 binds to LH/CG R, if binding is of high affinity, and if the receptor also binds arrestin3. Desensitization of intact LH/CG R was equally sensitive to ectopic constructs of arrestin2 that bind other G protein-coupled receptors (GPCRs) either in a phosphorylation-independent or -dependent manner. Intact LH/CG R was not desensitized by ectopic arrestin3 constructs. Surface plasmon resonance studies showed that arrestin2 bound a synthetic third intracellular (3i) LH/CG R loop peptide with picomolar affinity; arrestin3 bound with millimolar affinity. To determine whether Asp-564 in the 3i loop mimicked the phosphorylated residue of other GPCRs, human embryonic kidney (HEK) cells were transfected with wild-type (WT) and D564G LH/CG R. An agonist-stimulated ARF6-dependent arrestin2 undocking pathway to drive desensitization of WT receptor was recapitulated in HEK cell membranes, and ectopic arrestin2 promoted desensitization of WT LH/CG R. However, D564G LH/CG R in HEK cells was not desensitized, and synthetic 3i D564G peptide did not bind arrestin2. Synthetic 3i loop peptides containing D564E, D564V, or D564N also did not bind arrestin2. We conclude that the ARF6-mediated mechanism to release a pool of membrane-delimited arrestin to bind GPCRs may be a widespread mechanism to deliver arrestin to GPCRs for receptor desensitization. Unlike other GPCRs that additionally require receptor phosphorylation, LH/CG R activation is sufficient to expose a conformation in which Asp-564 in the 3i loop confers high affinity binding selectively to arrestin2.
- Délégation Ile-de-France Sud France
- University of Illinois at Chicago United States
- Vanderbilt University United States
- Northwestern University United States
- Research Triangle Park Foundation United States
570, ADP-Ribosylation Factors - metabolism, Arrestins, Protein Conformation, Swine, Molecular Sequence Data, LH - chemistry, 610, Transfection, Cell Line, Cyclic AMP - metabolism, LH - physiology, Structure-Activity Relationship, Ovarian Follicle, Receptors, Cyclic AMP, Animals, Humans, Amino Acid Sequence, Phosphorylation, Ovarian Follicle - metabolism, beta-Arrestins, Aspartic Acid, ADP-Ribosylation Factors, Electron Spin Resonance Spectroscopy, Arrestins - metabolism, Receptors, LH, ADP-Ribosylation Factor 6, Aspartic Acid - physiology, Female, Protein Binding
570, ADP-Ribosylation Factors - metabolism, Arrestins, Protein Conformation, Swine, Molecular Sequence Data, LH - chemistry, 610, Transfection, Cell Line, Cyclic AMP - metabolism, LH - physiology, Structure-Activity Relationship, Ovarian Follicle, Receptors, Cyclic AMP, Animals, Humans, Amino Acid Sequence, Phosphorylation, Ovarian Follicle - metabolism, beta-Arrestins, Aspartic Acid, ADP-Ribosylation Factors, Electron Spin Resonance Spectroscopy, Arrestins - metabolism, Receptors, LH, ADP-Ribosylation Factor 6, Aspartic Acid - physiology, Female, Protein Binding
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