K660 in the 3. extracellular loop of the TSH receptor is important for G-protein mediated signaling

OBJECTIVE: A new family of guanosine triphosphatase-activating proteins known as regulators of G protein signaling (RGS) has been found to regulate the desensitization of several G protein-coupled ligand-induced processes. The expression of nine RGS mRNAs was found in human thyroid tissue (RGS 2, 3, 5, 6, 9, 10, 12, 14 and 16). At present, little is known as to whether any of the RGS proteins play a role in TSH signaling. DESIGN AND METHODS: To explore the involvement of RGS proteins in the regulation of TSH receptor (TSHR) signal transduction, mRNA expression levels of the RGS proteins were analyzed after TSH stimulation of human thyroid primary cultures by real-time RT-PCR. Furthermore, the effects of RGS 2 expression on TSHR signaling (cAMP-, inositol-3-phosphate accumulation, TSHR cell surface expression) were studied in COS-7 cells. RESULTS: Only RGS 2 mRNA was found to be regulated by TSH in thyroid primary cultures. Co-expression of RGS 2 and TSHR in COS-7 cells reduced the TSHR signaling via inositol-3-phosphate but not via cAMP after stimulation with TSH. CONCLUSION: TSH-dependent RGS 2 mRNA expression and the suppression of TSH-G(q)alpha signaling by the overexpression of RGS 2 imply that RGS 2 is involved in TSHR-induced G(q) signal transduction.

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The Third Extracellular Loop of the β2-Adrenergic Receptor Can Modulate Receptor/G Protein Affinity

Molecular Pharmacology ◽

10.1124/mol.53.3.524 ◽

1998 ◽

Vol 53 (3) ◽

pp. 524-529 ◽

Cited By ~ 30

Author(s):

Ming-Ming Zhao ◽

Robert J. Gaivin ◽

Dianne M. Perez

Keyword(s):

G Protein ◽

Adrenergic Receptor ◽

Extracellular Loop ◽

The Third ◽

Protein Affinity ◽

Β2 Adrenergic Receptor

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N-linked glycosylation of protease-activated receptor-1 at extracellular loop 2 regulates G-protein signaling bias

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1508838112 ◽

2015 ◽

Vol 112 (27) ◽

pp. E3600-E3608 ◽

Cited By ~ 30

Author(s):

Antonio G. Soto ◽

Thomas H. Smith ◽

Buxin Chen ◽

Supriyo Bhattacharya ◽

Isabel Canto Cordova ◽

...

Keyword(s):

G Protein ◽

Fiber Formation ◽

G Protein Signaling ◽

Protein Signaling ◽

Extracellular Loop ◽

Glycoprotein Synthesis ◽

Mutant Receptor ◽

Rhoa Activation ◽

Protease Activated Receptor 1 ◽

Loop 2

Protease-activated receptor-1 (PAR1) is a G-protein-coupled receptor (GPCR) for the coagulant protease thrombin. Similar to other GPCRs, PAR1 is promiscuous and couples to multiple heterotrimeric G-protein subtypes in the same cell and promotes diverse cellular responses. The molecular mechanism by which activation of a given GPCR with the same ligand permits coupling to multiple G-protein subtypes is unclear. Here, we report that N-linked glycosylation of PAR1 at extracellular loop 2 (ECL2) controls G12/13 versus Gq coupling specificity in response to thrombin stimulation. A PAR1 mutant deficient in glycosylation at ECL2 was more effective at stimulating Gq-mediated phosphoinositide signaling compared with glycosylated wildtype receptor. In contrast, wildtype PAR1 displayed a greater efficacy at G12/13-dependent RhoA activation compared with mutant receptor lacking glycosylation at ECL2. Endogenous PAR1 rendered deficient in glycosylation using tunicamycin, a glycoprotein synthesis inhibitor, also exhibited increased PI signaling and diminished RhoA activation opposite to native receptor. Remarkably, PAR1 wildtype and glycosylation-deficient mutant were equally effective at coupling to Gi and β-arrestin-1. Consistent with preferential G12/13 coupling, thrombin-stimulated PAR1 wildtype strongly induced RhoA-mediated stress fiber formation compared with mutant receptor. In striking contrast, glycosylation-deficient PAR1 was more effective at increasing cellular proliferation, associated with Gq signaling, than wildtype receptor. These studies suggest that N-linked glycosylation at ECL2 contributes to the stabilization of an active PAR1 state that preferentially couples to G12/13 versus Gq and defines a previously unidentified function for N-linked glycosylation of GPCRs in regulating G-protein signaling bias.

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