The Role of Subtype-specific Ligand Binding and the C-tail Domain in Dimer Formation of Human Somatostatin Receptors

Michael Grant,Ramesh C Patel,Ujendra Kumar

doi:10.1074/jbc.m406276200

Abstract

G-protein-coupled receptors (GPCRs) represent the largest and most diverse family of cell surface receptors. Several GPCRs have been documented to dimerize with resulting changes in pharmacology. We have previously reported by means of photobleaching fluorescence resonance energy transfer (pbFRET) microscopy and fluorescence correlation spectroscopic (FCS) analysis in live cells, that human somatostatin receptor (hSSTR) 5 could both homodimerize and heterodimerize with hSSTR1 in the presence of the agonist SST-14. In contrast, hSSTR1 remained monomeric when expressed alone regardless of agonist exposure in live cells. In an effort to elucidate the role of ligand and receptor subtypes in heterodimerization, we have employed both pb-FRET microscopy and Western blot on cells stably co-expressing hSSTR1 and hSSTR5 treated with subtype-specific agonists. Here we provide evidence that activation of hSSTR5 but not hSSTR1 is necessary for heterodimeric assembly. This property was also reflected in signaling as shown by increases in adenylyl cyclase coupling efficiencies. Furthermore, receptor C-tail chimeras allowed for the identification of the C-tail as a determinant for dimerization. Finally, we demonstrate that heterodimerization is subtype-selective involving ligand-induced conformational changes in hSSTR5 but not hSSTR1 and could be attributed to molecular events occurring at the C-tail. Understanding the mechanisms by which GPCRs dimerize holds promise for improvements in drug design and efficacy.

Highlights

G-protein-coupled receptors (GPCRs) represent the largest and most diverse family of cell surface receptors
We have previously reported by means of photobleaching fluorescence resonance energy transfer microscopy and fluorescence correlation spectroscopic (FCS) analysis in live cells, that human somatostatin receptor 5 could both homodimerize and heterodimerize with hSSTR1 in the presence of the agonist SST-14
To determine the physical association between the two receptors, we performed photobleaching fluorescence resonance energy transfer (pbFRET) microscopy on the cells by using a primary antibody followed by a secondary antibody conjugated with fluorescein to hSSTR1 and an anti-HA monoclonal antibody conjugated with rhodamine to hSSTR5

Summary

EXPERIMENTAL PROCEDURES

Materials and Antisera—The peptides SST-14, D-Trp-SST-14, SST28, and [Leu (8)-D-Trp-22, Tyr-25]-SST-28 (LTT-SST-28) were purchased from Bachem, Torrance, CA; Octreotide [SMS (201-995)] was given by Sandoz, Basel, Switzerland and des-AA1,2,5-[D-Trp8IAmp9]SS (SCH-275) was a gift from Dr J. Binding studies were performed with 20 – 40 ␮g of membrane protein collected from CHO-K1 cells stably expressing the receptor constructs, and 125I-labeled LTT-SST-28 radioligand (ϳ60 pM) in 50 mM HEPES, pH 7.5, 2 mM CaCl2, 5 mM MgCl2, 0.5% bovine serum albumin, 0.02% phenylmethylsulfonyl fluoride, and 0.02% bacitracin (binding buffer) for 30 min at 37 °C. PbFRET Microscopy and Immunocytochemistry—PbFRET experiments were performed on CHO-K1 cells as previously described (13, 14, 29, 33) stably co-expressing HA-hSSTR5 and hSSTR1, and mono-expressing hSSTR5, hSSTR1, and the receptor chimeras. Co-immunoprecipitation and Western Blot—Membranes from HAhSSTR1, HA-SSTR5, and HA-hSSTR1/c-Myc-hSSTR5 stably transfected in HEK-293 cells were prepared using a glass homogenizer in 20 mM Tris-HCl, 2.5 mM dithiothreitol, pH 7.5 as previously described (13). Images were captured using an Alpha Innotech FluorChem 8800 (Alpha Innotech Co., San Leandro, CA) gel box imager and densitometry was carried out using FluorChem software (Alpha Innotech Co.)

RESULTS

TABLE II Comparison of adenylyl cyclase coupling efficiencies by SST agonists

DISCUSSION