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Heterodimerization of somatostatin and opioid receptors cross-modulates phosphorylation, internalization, and
Manuela Pfeiffer1, Thomas Koch, Helmut Schröder
1Department of Pharmacology and Toxicology, Otto-von-Guericke University, 39120 Magdeburg, Germany.
Abstract:
Heterodimerization has been shown to modulate the ligand binding, signaling, and trafficking properties of G protein-coupled receptors. However, to what extent heterodimerization may alter agonist-induced phosphorylation and desensitization of these receptors has not been documented. We have recently shown that heterodimerization of sst(2A) and sst(3) somatostatin receptors results in inactivation of sst(3) receptor function (Pfeiffer, M., Koch, T., Schröder, H., Klutzny, M., Kirscht, S., Kreienkamp, H. J., Höllt, V., and Schulz, S. (2001) J. Biol. Chem. 276, 14027-14036). Here we examine dimerization of the sst(2A) somatostatin receptor and the mu-opioid receptor, members of closely related G protein-coupled receptor families. In coimmunoprecipitation studies using differentially epitope-tagged receptors, we provide direct evidence for heterodimerization of sst(2A) and MOR1 in human embryonic kidney 293 cells. Unlike heteromeric assembly of sst(2A) and sst(3), sst(2A)-MOR1 heterodimerization did not substantially alter the ligand binding or coupling properties of these receptors. However, exposure of the sst(2A)-MOR1 heterodimer to the sst(2A)-selective ligand L-779,976 induced phosphorylation, internalization, and desensitization of sst(2A) as well as MOR1. Similarly, exposure of the sst(2A)-MOR1 heterodimer to the mu-selective ligand [d-Ala(2),Me-Phe(4),Gly(5)-ol]enkephalin induced phosphorylation and desensitization of both MOR1 and sst(2A) but not internalization of sst(2A). Cross-phosphorylation and cross-desensitization of the sst(2A)-MOR1 heterodimer were selective; they were neither observed with the sst(2A)-sst(3) heterodimer nor with the endogenously expressed lysophosphatidic acid receptor. Heterodimerization may thus represent a novel regulatory mechanism that could either restrict or enhance phosphorylation and desensitization of G protein-coupled receptors.
Insights
G protein-coupled receptor heterodimerization, specifically between somatostatin receptor subtype 2A (sst(2A)) and the mu-opioid receptor (MOR1), leads to cross-phosphorylation and cross-desensitization. This novel regulatory mechanism impacts receptor function upon agonist exposure.
Area of Science:
- Molecular and Cellular Biology
- Pharmacology
- Biochemistry
Background:
- G protein-coupled receptors (GPCRs) form heterodimers, influencing their ligand binding, signaling, and trafficking.
- The impact of heterodimerization on agonist-induced phosphorylation and desensitization of GPCRs remains largely undocumented.
- Previous studies showed heterodimerization of sst(2A) and sst(3) somatostatin receptors leads to sst(3) functional inactivation.
Purpose of the Study:
- To investigate the heterodimerization of the sst(2A) somatostatin receptor and the mu-opioid receptor (MOR1).
- To determine if sst(2A)-MOR1 heterodimerization alters ligand binding, coupling, phosphorylation, or desensitization.
- To explore the phenomenon of cross-phosphorylation and cross-desensitization in GPCR heterodimers.
Main Methods:
- Coimmunoprecipitation studies using differentially epitope-tagged sst(2A) and MOR1 receptors in human embryonic kidney 293 cells.
- Assessment of ligand binding and G protein coupling properties of the sst(2A)-MOR1 heterodimer.
- Analysis of agonist-induced phosphorylation, internalization, and desensitization of both receptors within the heterodimer.
Main Results:
- Direct evidence for sst(2A)-MOR1 heterodimerization was established.
- Unlike sst(2A)-sst(3) heterodimers, sst(2A)-MOR1 heterodimerization did not significantly alter ligand binding or coupling.
- Agonist stimulation of the sst(2A)-MOR1 heterodimer induced phosphorylation and desensitization of both sst(2A) and MOR1, demonstrating cross-phosphorylation and cross-desensitization.
Conclusions:
- GPCR heterodimerization, exemplified by sst(2A)-MOR1, can lead to cross-phosphorylation and cross-desensitization.
- This cross-regulation is selective and was not observed with sst(2A)-sst(3) heterodimers or endogenous lysophosphatidic acid receptors.
- GPCR heterodimerization represents a novel regulatory mechanism with the potential to modulate receptor phosphorylation and desensitization.