Homo- and Heterodimerization of Somatostatin Receptor Subtypes: INACTIVATION OF sst3 RECEPTOR FUNCTION BY HETERODIMERIZATION WITH sst2A

Manuela Pfeiffer,Thomas Koch,Helmut Schröder,Marcus Klutzny,Susanne Kirscht,Hans-Jürgen Kreienkamp,Volker Höllt,Stefan Schulz

doi:10.1074/jbc.m006084200

Abstract

Several recent studies suggest that G protein-coupled receptors can assemble as heterodimers or hetero-oligomers with enhanced functional activity. However, inactivation of a fully functional receptor by heterodimerization has not been documented. Here we show that the somatostatin receptor (sst) subtypes sst(2A) and sst(3) exist as homodimers at the plasma membrane when expressed in human embryonic kidney 293 cells. Moreover, in coimmunoprecipitation studies using differentially epitope-tagged receptors, we provide direct evidence for heterodimerization of sst(2A) and sst(3). The sst(2A)-sst(3) heterodimer exhibited high affinity binding to somatostatin-14 and the sst(2)-selective ligand L-779,976 but not to the sst(3)-selective ligand L-796,778. Like the sst(2A) homodimer, the sst(2A)-sst(3) heterodimer stimulated guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding, inhibition of adenylyl cyclase, and activation of extracellular signal-regulated kinases after exposure to the sst(2)-selective ligand L-779,976. However, unlike the sst(3) homodimer, the sst(2A)-sst(3) heterodimer did not promote GTPgammaS binding, adenylyl cyclase inhibition, or extracellular signal-regulated kinase activation in the presence of the sst(3)-selective ligand L-796,778. Interestingly, during prolonged somatostatin-14 exposure, the sst(2A)-sst(3) heterodimer desensitized at a slower rate than the sst(2A) and sst(3) homodimers. Both sst(2A) and sst(3) homodimers underwent agonist-induced endocytosis in the presence of somatostatin-14. In contrast, the sst(2A)-sst(3) heterodimer separated at the plasma membrane, and only sst(2A) but not sst(3) underwent agonist-induced endocytosis after exposure to somatostatin-14. Together, heterodimerization of sst(2A) and sst(3) results in a new receptor with a pharmacological and functional profile resembling that of the sst(2A) receptor, however with a greater resistance to agonist-induced desensitization. Thus, inactivation of sst(3) receptor function by heterodimerization with sst(2A) or possibly other G protein-coupled receptors may explain some of the difficulties in detecting sst(3)-specific binding and signaling in mammalian tissues.

Highlights

Several recent studies suggest that G protein-coupled receptors can assemble as heterodimers or hetero-oligomers with enhanced functional activity
We show that the somatostatin receptor subtypes sst2A and sst3 exist as homodimers at the plasma membrane when expressed in human embryonic kidney 293 cells
The actions of SS-14 are mediated via five distinct somatostatin receptor subtypes, termed sst1–sst5, which belong to the superfamily of G protein-coupled receptors (GPCRs)

Summary

Introduction

Several recent studies suggest that G protein-coupled receptors can assemble as heterodimers or hetero-oligomers with enhanced functional activity. It should be noted that the higher levels of total somatostatin receptor proteins (sst2A plus sst3) present in coexpressing cells were not associated with an equivalent increase in the total number of somatostatin binding sites detected in saturation binding assays (Bmax for T7sst2A-Mycsst3 1088 Ϯ 66 fmol/mg as compared with Bmax for T7sst2A 820 Ϯ 19 fmol/mg and Bmax for T7sst3 1182 Ϯ 31 fmol/mg).

Results

Conclusion