Evidence of G-protein-coupled receptor and substrate transporter heteromerization at a single molecule level

Abstract

G-protein-coupled receptors (GPCRs) can constitute complexes with non-GPCR integral membrane proteins, while such interaction has not been demonstrated at a single molecule level so far. We here investigated the potential interaction between the thyrotropin receptor (TSHR) and the monocarboxylate transporter 8 (MCT8), a member of the major facilitator superfamily (MFS), using fluorescence cross-correlation spectroscopy (FCCS). Both the proteins are expressed endogenously on the basolateral plasma membrane of the thyrocytes and are involved in stimulation of thyroid hormone production and release. Indeed, we demonstrate strong interaction between both the proteins which causes a suppressed activation of G by TSH-stimulated TSHR. Thus, we provide not only evidence for a novel interaction between the TSHR and MCT8, but could also prove this interaction on a single molecule level. Moreover, this interaction forces biased signaling at the TSHR. These results are of general interest for both the GPCR and the MFS research fields.

Evaluation of a rare glucose‐dependent insulinotropic polypeptide receptor variant in a patient with diabetes

“…For HA‐tag detection, cells were washed, fixed with paraformaldehyde and probed with a biotin‐labeled anti‐HA antibody (Roche Applied Science, Mannheim, Germany). The bound anti‐HA‐biotin antibody was detected using peroxidase‐labeled streptavidin (BioLegend, London, UK) in a substrate/chromogen reaction as previously described . Eight additional glycine residues (GIPR‐WT 8xGly) were inserted directly after the HA tag to increase flexibility and facilitate proper detection of cell surface expression of GIPR.…”

“…Cells were stimulated for 45 minutes with GIP (decadic concentration‐response curves from 1 pM to 1 μM) in stimulation buffer containing 138 mM NaCl, 6 mM KCl, 1 mM MgCl 2 , 5.5 mM glucose, 20 mM HEPES, 1 mM CaCl 2 and 1 mM 3‐isobutyl‐1‐methylxanthine (IBMX, Sigma Aldrich, St. Louis, Missouri). cAMP accumulation was determined using AlphaScreen technology (Perkin Elmer, Life Science, Zaventem, Belgium) as previously described …”

“…The bound anti-HA-biotin antibody was detected using peroxidase-labeled streptavidin (BioLegend, London, UK) in a substrate/chromogen reaction as previously described. 14 Eight additional glycine residues (GIPR-WT 8xGly) were inserted directly after the HA tag to increase flexibility and facilitate proper detection of cell surface expression of GIPR. This linker was also introduced in all of the examined GIPR variants.…”

“…cAMP accumulation was determined using AlphaScreen technology (Perkin Elmer, Life Science, Zaventem, Belgium) as previously described. 14…”

Constitutive TSH receptor activation as a hallmark of thyroid autonomy

“…antibody driven TSH receptor activation in Graves' disease and ophthalmopathy [21,22]. As another example, it was recently demonstrated that the TSHR can also form complexes with other non-GPCR membrane proteins such as the mono-carboxylate transporter 8, which expressed on the basolateral membrane of thyrocytes is involved in thyroid hormone release [23]. This hints at truly complex regulation of thyroid hormone production at the level of the thyroid gland and takes the thinking from an individual receptor to the broader and hitherto understudied consideration of interacting protein networks in the thyrocyte membrane, which may be relevant for a better understanding of thyroid disease.…”