Heterodimerization of Somatostatin and Opioid Receptors Cross-modulates Phosphorylation, Internalization, and Desensitization

Abstract

Recent biochemical, biophysical, and functional studies suggest that G protein-coupled receptors (GPCRs) 1 can assemble as homo-or heterodimeric complexes (1, 2). Heterodimerization has been shown to alter both ligand binding affinity and signaling efficacy of GPCRs (1, 2). ␦-and -opioid receptors form stable heterodimers with ligand binding and signaling properties resembling that of the 2 receptor (3). Formation of heterodimers between the sst 1 and sst 5 somatostatin receptors has been found to modulate the pharmacology and signaling of both receptors (4). The ␥-aminobutyric acid receptor B is unique in that heterodimerization of the nonfunctional ␥-aminobutyric acid receptors B1 and B2 is required for native affinity for ligands and complete functional activity (5-9). Heteromeric assembly of fully functional AT 1 angiotensin II and B 2 bradykinin receptors results in increased efficacy of angiotensin II and decreased efficacy of bradykinin (10). Heterodimerization has also been shown to alter endocytotic trafficking of GPCRs (3, 4, 10, 11). The -␦ heterodimer exhibited a decrease in agonist-mediated receptor endocytosis (3). Oligomerization of ␦-and -opioid receptors with the distantly related ␤ 2 -adrenergic receptor results in increased and decreased receptor endocytosis, respectively (11). AT 1 -B 2 heterodimerization induced a switch to a clathrin-and dynamindependent endocytotic pathway for both receptors (10). Signaling of GPCRs is often terminated by phosphorylation of intracellular serine and threonine residues. After phosphorylation of the receptor, arrestins are frequently recruited to the plasma membrane, at which they facilitate endocytosis by serving as scaffolding proteins that bind to clathrin. Although changes in trafficking have been clearly documented, agonistinduced phosphorylation and desensitization of these GPCR heterodimers has not been examined.We have recently shown that the sst 2A and sst 3 somatostatin receptors exist as constitutive homodimers when expressed alone and as constitutive heterodimers when coexpressed in human embryonic kidney (HEK) 293 cells (12). Whereas the sst 2A -sst 3 heterodimer behaved like the sst 2A homodimer, it did not reproduce the pharmacological characteristics of the sst 3 homodimer, suggesting that physical interaction of sst 3 with sst 2A induced functional inactivation of the sst 3 subtype (12). Here we report that the sst 2A receptor also forms stable heterodimers with the -opioid receptor (MOR1), a member of a closely related GPCR family. Unlike that observed for the sst 2A -sst 3 heterodimer, sst 2A -MOR1 heterodimerization did not significantly affect the ligand binding or coupling properties but promoted cross-modulation of phosphorylation, internalization, and desensitization of these receptors. EXPERIMENTAL PROCEDURES Materials

Dimerization of the Human Receptors for Prostacyclin and Thromboxane Facilitates Thromboxane Receptor-mediated cAMP Generation

“…Whereas the presence of IP/TP␣ heterodimers in IP/TP␣-HEK 293 cells does not constitute definitive proof of their involvement in the alterations in TP␣ signaling, the dependence of TP␣-mediated cAMP formation on the physical presence of IP, in overexpressing and native cell models, strongly suggests biologically relevant heterodimerization between these two receptors. In addition, data indicating the formation of an alternative ligand binding site (see below), a frequent consequence of GPCR dimerization (22,29,30), is consistent with the formation of an IP/TP␣ complex, as is the synergistic augmentation of TP-cAMP generation following preactivation of IP (22).…”

“…Indeed, a number of studies investigating the signaling properties of newly discovered GPCR heterodimers describe an altered receptorligand-effector profile (22,29,30). Using SQ 29548 to label the TP␣ and iloprost to label the IP, we found no alterations in the binding affinities of specific IP or TP ligands to their individual receptor sites, suggesting preservation of the binding characteristics (Table I).…”

“…Receptor-Ligand Interactions-It has been demonstrated previously that receptor heterodimerization can alter the affinity of the individual receptors for their specific ligands (22,29,30). We examined if changes in ligand binding in the IP/TP␣ heterodimer could underlie the changes we observed in TP␣ signaling.…”

Heterodimerization of the α and β isoforms of the human thromboxane receptor enhances isoprostane signaling

“…One of the characteristics frequently modified as a result of GPCR heterodimerization is the affinity of the individual receptors for their respective ligands [15,16]. We examined if altered ligand binding to the TPα/TPβ heterodimer might underlie the changes we observed in TPsignaling.…”

Subunits of a Yeast Oligomeric G Protein-coupled Receptor Are Activated Independently by Agonist but Function in Concert to Activate G Protein Heterotrimers

“…In one model, agonist binding to one receptor subunit induces a conformational change that is transmitted allosterically to another receptor subunit, which in turn undergoes a conformational change resulting in G protein activation. This mechanism has been suggested by studies of the GABA(B) receptor heterodimers (16 -20), by cooperative agonist binding to receptors such as muscarinic acetylcholine receptors (21,22), and by studies indicating that heterodimerization can alter the signaling, trafficking, or desensitization of opioid, ␤-adrenergic, bradykinin, or angiotensin II receptors (23)(24)(25)(26)(27). In a second model suggested by studies of GABA(B) receptors (16 -20), agonist binding to the R1 subunit induces movement that relieves its inhibitory action on the R2 subunit, which is freed for G protein activation.…”