Aspartic acid 564 in the third cytoplasmic loop of the luteinizing hormone/choriogonadotropin receptor is crucial for phosphorylation-independent interaction with arrestin2

Sutapa Mukherjee; Vsevolod V Gurevich; Anita Preninger; Heidi E Hamm; Marie-France Bader; Asgerally T Fazleabas; Lutz Birnbaumer; Mary Hunzicker-Dunn

doi:10.1074/jbc.M110479200

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Aspartic acid 564 in the third cytoplasmic loop of the luteinizing hormone/choriogonadotropin receptor is crucial for phosphorylation-independent interaction with arrestin2

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Aspartic acid 564 in the third cytoplasmic loop of the luteinizing hormone/choriogonadotropin receptor is crucial for phosphorylation-independent interaction with arrestin2

Sutapa Mukherjee, Vsevolod V Gurevich, Anita Preninger, Heidi E Hamm, Marie-France Bader, Asgerally T Fazleabas, Lutz Birnbaumer and Mary Hunzicker-Dunn

The Journal of biological chemistry, Vol.277(20), pp.17916-17927

05/17/2002

DOI: https://doi.org/10.1074/jbc.M110479200

Handle:

https://hdl.handle.net/2376/114498

PMID: 11867621

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Abstract

Amino Acid Sequence

Cell Line

Receptors, LH - chemistry

Phosphorylation

Electron Spin Resonance Spectroscopy

Humans

Molecular Sequence Data

Structure-Activity Relationship

Arrestins - metabolism

Animals

Transfection

Swine

beta-Arrestins

Protein Binding

Female

Protein Conformation

ADP-Ribosylation Factors - metabolism

Aspartic Acid - physiology

Cyclic AMP - metabolism

Ovarian Follicle - metabolism

Receptors, LH - physiology

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.

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Title: Aspartic acid 564 in the third cytoplasmic loop of the luteinizing hormone/choriogonadotropin receptor is crucial for phosphorylation-independent interaction with arrestin2
Creators: Sutapa Mukherjee - Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611, USA
Vsevolod V Gurevich
Anita Preninger
Heidi E Hamm
Marie-France Bader
Asgerally T Fazleabas
Lutz Birnbaumer
Mary Hunzicker-Dunn
Publication Details: The Journal of biological chemistry, Vol.277(20), pp.17916-17927
Academic Unit: Molecular Biosciences, School of
Publisher: United States
Grant note: R01 EY 11500 / NEI NIH HHS EY 06062 / NEI NIH HHS GM 63097 / NIGMS NIH HHS R01 HD 38060 / NICHD NIH HHS R01 EY 10291 / NEI NIH HHS
Identifiers: 99900547739801842
Language: English
Resource Type: Journal article