Selective Regulation of G Protein-Coupled Receptor-Mediated Signaling by G Protein-Coupled Receptor Kinase 2 in FRTL-5 Cells: Analysis of Thyrotropin, α1B-Adrenergic, and A1 Adenosine Receptor-Mediated Responses
- Consorzio Mario Negri Sud, Istituto di Ricerche Farmacologiche “Mario Negri,” Santa Maria Imbaro, Italy
Abstract
G protein-coupled receptor kinases (GRKs) play a key role in the process of receptor homologous desensitization. In the present study, we address the question of whether a variety of receptors coupled to different G protein subtypes and naturally expressed on the same cell are selectively regulated by GRK2. The signaling stimulated by thyrotropin (TSH), α1B-adrenergic, and A1adenosine receptors was studied in FRTL-5 cells permanently transfected to overexpress GRK2 and GRK2-K220R, a kinase dead GRK dominant negative mutant. In FRTL-5 overexpressing GRK2, TSH-induced cyclic AMP response was attenuated, indicating that TSH receptor is desensitized by this kinase. Consistently, FRTL-5 cells overexpressing GRK2-K220R show increased TSH-induced cyclic AMP response, demonstrating that this receptor is under tonic control by GRK. Unlike TSH receptor, α1B-adrenergic receptor response was unaffected in FRTL-5 overexpressing GRK2 and GRK2-K220R. When A1 adenosine receptors were stimulated, Giα-mediated cyclic AMP inhibition was totally unaffected by overexpression of either GRK2 or GRK2-K220R. By contrast, Gβγ-mediated response (activation of mitogen-activated protein kinases) was efficiently desensitized by GRK2 but was unaffected by GRK2-K220R overexpression. The present study documents that overexpression of GRK2 results in a selective regulation of different G protein-coupled receptors expressed on the same cell and that this kinase can regulate preferentially only one of the different pathways activated by the same receptor. The preferential regulation of the A1 adenosine receptor-stimulated mitogen-activated protein kinases by GRK2 indicates that this kinase can have additional regulatory effects on Gβγ-stimulated pathways, possibly through direct binding and regulation of the receptor-Gβγ complex.
Footnotes
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Send reprint requests to: Dr. Luisa Iacovelli, Consorzio Mario Negri Sud, via Nazionale 66030, S. Maria Imbaro, Italy. E-mail:iacovell{at}cmns.mnegri.it
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This work was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC), by Consiglio Nazionale delle Ricerche (CNR) Target Project on Biotechnology, and by European Community Biomed 2 program-PL 963566. R.F. was the recipient of a fellowship granted by Progetto Speciale Ricerca Scientifica e Applicata nel Mezzogiorno PS35-93/IND.
- Abbreviations:
- GPCR
- G protein-coupled receptor
- FSK
- forskolin
- A1r
- A1 adenosine receptor(s)
- CaM
- calmodulin
- CPA
- N6-cyclopentyladenosine
- HEK
- human embryonic kidney
- cAMP
- cyclic AMP
- HBSS
- Hanks’ balanced salt solution
- ERK
- extracellular signal-regulated kinase(s)
- MAPK
- mitogen-activated protein kinase
- TSH
- thyrotropin
- TSHr
- thyrotropin receptor(s)
- NE
- norepinephrine
- IP
- inositol phosphate(s)
- GRK
- G protein-coupled receptor kinase
- PC
- phosphatidylcholine
- ROS
- rod outer segment(s)
- PIP2
- phosphatidyl inositol-4,5-bisphosphate
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- Received December 28, 1998.
- Accepted April 30, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
