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. 2008 Jan;22(1):126-38.
doi: 10.1210/me.2007-0352. Epub 2007 Sep 13.

Contributions of intracellular loops 2 and 3 of the lutropin receptor in Gs coupling

Krassimira Angelova  1 , Francesca Fanelli, David Puett
Affiliations

Contributions of intracellular loops 2 and 3 of the lutropin receptor in Gs coupling

Krassimira Angelova et al. Mol Endocrinol. 2008 Jan.

Abstract

A number of amino acids essential for Gs coupling, i.e. hot spots, were identified after in vitro Ala-scanning mutagenesis of the cytosolic extensions of helices 3, 5, and 6 and of intracellular loops 2 and 3 (IL2 and IL3) of the human LH receptor (LHR). Consistent with the results of in vitro experiments involving ligand binding and ligand-mediated signaling in transiently transfected human embryonic kidney 293 cells, computational modeling of the isolated receptor and of the receptor-G protein complexes suggests an important role of the cytosolic extension of helix 3 and the N-terminal portion of the IL2 in Gs(alpha) interaction, whereas the contribution of IL3 is marginal. Mapping the hot spots into the computational models of LHR and the LHR-Gs complexes allowed for a distinction between receptor sites required for intramolecular structural changes (i.e. I460, T461, H466, and I549) and receptor sites more likely involved in G protein recognition (i.e. R464, T467, I468, Y470, Y550, and D564). The latter sites include the highly conserved arginine of the (E/D)R(Y/W) motif, which is therefore likely to be a receptor recognition point for Gs rather than a switch of receptor activation. The results of in vitro and in silico experiments carried out in this study represent the first comprehensive delineation of functionality of the individual residues in the intracellular domains of LHR and establish potential switches of receptor activation as well as a map of the primary receptor recognition sites for Gs. A novel way to consider constitutively active mutants was inferred from this study, i.e. receptor states with improved complementarity for the G protein compared to the wild-type receptor.

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Figures

Figure 1
Figure 1
Rhodopsin and Human LHR Sequence Alignment Sequence alignment between bovine rhodopsin [PDB code: 1U19 (27)], i.e. template, and the human LHR (target) that was employed for comparative modeling. The amino acid stretches 100–101, 106–107, and 236–242 have been deleted from the 1U19 template. The boxed amino acid in each rhodopsin helix corresponds to the amino acid n.50 according to the Ballesteros and Weinstein nomenclature (17), whereas the boxed amino acid stretch at the end of H7 corresponds to H8. N-TER, N-terminal.
Figure 2
Figure 2
Complex between Gs and D564(6.30)G LHR CAM Left, Side view in a direction parallel to the membrane plane of a selected complex between a AVG1000ps-minimized structure of LHR (green) and Gsαβγ. The Gs α-, β-, and γ-subunits are violet, orange, and blue, respectively. Right, The cytosolic half of the D564(6.30)G LHR CAM and selected domains of Gsα are shown. The complex is seen from the intracellular side in a direction perpendicular to the membrane surface. The amino acid side chains involved in the most recurrent interactions are represented by sticks. Gsα is violet, whereas the receptor domains are colored as follows: H1, H2, H3, H4, H5, H6, and H7 are in blue, orange, green, pink, yellow, cyan, and purple, respectively, whereas IL1, IL2, and IL3 are lime, slate, and magenta, respectively.
Figure 3
Figure 3
Representative Competition Binding and cAMP Dose-Response Curves for WT LHR and I460(3.46)A, T461(3.47)A, R464(3.50)A, and T467(3.53)A LHR Mutants A, Competitive binding of the WT and mutant LHRs. HEK 293 cells were transiently transfected with the various constructs and 48 h later characterized for hCG binding. The binding experiments were performed in Waymouth’s medium/BSA for 6 h at 37 C in the presence of 50 pm [125I]hCG and various concentrations of hCG. B, Concentration-dependent hCG-stimulated cAMP accumulation in HEK 293 cells expressing the WT and mutant LHRs. The transfected cells were stimulated with increasing concentrations of hCG for 30 min at 37 C in the presence of Waymouth’s medium/BSA and 0.8 mm isobutylmethylxanythine.
Figure 4
Figure 4
Coupling Efficiencies of Ala-Substituted Residues in IL2 of LHR From measurements of expression levels and of binding and signaling parameters, a coupling efficiency, Q, was determined for each Ala-substituted side chain in IL2. *, Significantly different (P ≤ 0.05) from that of WT LHR (Q = 1.00).
Figure 5
Figure 5
Coupling Efficiencies of LHR Ala Mutants in IL3 From measurements of expression levels and of binding and signaling parameters, Q was determined for each Ala-substituted side chain in IL3. *, Significantly different (P ≤ 0.05) from that of WT LHR (Q = 1.00).
Figure 6
Figure 6
Map of the Hot Spots in LHR Selected AVG1000ps -minimized structure of WT LHR seen from the intracellular side in a direction perpendicular to the membrane surface. The hot spots resulting from in vitro Ala scanning mutagenesis are highlighted by spheres centered at the Cα atoms. The extracellular domains are not shown. The receptor domains are colored as follows: H1, H2, H3, H4, H5, H6, and H7 are in blue, orange, green, pink, yellow, cyan, and violet, respectively, whereas IL1, IL2, and IL3 are lime, slate, and magenta, respectively.
Figure 7
Figure 7
Docking Outputs Concerning Different Forms of the LHR and Selected Amino Acids from the Receptor (black) and from the G Protein Involved in Charge-Reinforced H-Bonds (bold) or van der Waals Interactions (underlined)
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