Site‐Directed Mutations in the Third Intracellular Loop of the Serotonin 5‐HT1A Receptor Alter G Protein Coupling from Gi to Gs in a Ligand‐Dependent Manner

Malmberg, Åsa; Strange, Philip G.

doi:10.1046/j.1471-4159.2000.751283.x

Cited by 54 publications

(28 citation statements)

References 20 publications

(28 reference statements)

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“…Likewise, 5-HT1A-i2 loop peptides mediate direct coupling to inhibition of cAMP (Varrault et al, 1994). Mutation of Ci3 residues (V344E and T343A/V344E) enhanced receptor coupling to G s over G i , consistent with its role in determining G␣ i specificity (Malmberg and Strange, 2000). Thus, whereas Ci3 seems to mainly dictate G␣ i specificity, our results indicate that Ci2 is mainly implicated in G␤␥ signaling but also contributes to G␣ i signaling.…”

Section: Discussionsupporting

confidence: 80%

“…Thus, coactivation of G s by D1 receptor activation was required to observe DPAT-induced potentiation. This enhancement of D1 response was blocked by pertussis toxin treatment, indicating that the potentiation is mediated by G i /G o proteins and is not due to weak agonist-mediated stimulation of G s , as observed for mutants of the 5-HT1A Ci3 domain (Malmberg and Strange, 2000). DPAT-induced potentiation was greatest for the P150T-5-HT1A receptor, which lacked coupling to adenylyl cyclase II.…”

Section: Substitutionmentioning

confidence: 67%

“…Peptides corresponding to the 5-HT1A Ci3 domain mimic G␣ i -mediated inhibition of forskolin-stimulated AC and attenuate inhibitory coupling of 5-HT1A receptors to AC (Malmberg and Strange, 2000;Ortiz et al, 2000). Likewise, 5-HT1A-i2 loop peptides mediate direct coupling to inhibition of cAMP (Varrault et al, 1994).…”

Section: Discussionmentioning

confidence: 99%

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Molecular Determinants in the Second Intracellular Loop of the 5-Hydroxytryptamine-1A Receptor for G-Protein Coupling

et al. 2006

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This study provides the first comprehensive evidence that the second intracellular loop C-terminal domain (Ci2) is critical for receptor-G protein coupling to multiple responses. Although Ci2 is weakly conserved, its role in 5-hydroxytryptamine-1A (5-HT1A) receptor function was suggested by the selective loss of G␤␥-mediated signaling in the T149A-5-HT1A receptor mutant. More than 60 point mutant 5-HT1A receptors in the ␣-helical Ci2 sequence ( 143 DYVNKRTPRR 152 ) were generated. Most mutants retained agonist binding and were tested for G␤␥ signaling to adenylyl cyclase II or phospholipase C and G␣i coupling to detect constitutive and agonist-induced G i /G o coupling. Remarkably, most point mutations markedly attenuated 5-HT1A signaling, indicating that the entire Ci2 domain is critical for receptor G-protein coupling. Six signaling phenotypes were observed: wild-type-like, G␣ i -coupled/weak G␤␥-coupled, G␤␥-uncoupled, G␤␥-selective coupled, uncoupled, and inverse coupling. Our data elucidate specific roles of Ci2 residues consistent with predictions based on rhodopsin crystal structure. The absolute coupling requirement for lysine, arginine, and proline residues is consistent with a predicted amphipathic ␣-helical Ci2 domain that is kinked at Pro150. Polar residues (Thr149, Asn146) located in the externally oriented positively charged face were required for G␤␥ but not G␣ i coupling, suggesting a direct interface with G␤␥ subunits. The hydrophobic face includes the critical Tyr144 that directs the specificity of coupling to both G␤␥ and G␣ i pathways. The key coupling residues Tyr144/Lys147 (Ci2) are predicted to orient internally, forming hydrogen and ionic bonds with Asp133/ Arg134 (Ni2 DRY motif) and Glu340 (Ci3) to stabilize the Gprotein coupling domain. Thus, the 5-HT1A receptor Ci2 domain determines G␤␥ specificity and stabilizes G␣ i -mediated signaling.

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Section: Discussionsupporting

confidence: 80%

Section: Substitutionmentioning

confidence: 67%

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Molecular Determinants in the Second Intracellular Loop of the 5-Hydroxytryptamine-1A Receptor for G-Protein Coupling

et al. 2006

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“…Then a putative G protein-binding site is clearly shown by an arrangement, in which the iLP1 ( Figures 6A and B (red)) is located above the iLP3 (Figures 6and B (blue)), which forms a bed, and the iLP2 (Figures 6 A and B (yellow)) is set towards the back of the proposed pocket ( Figure 6C). This pocket, which is formed by the three loops, is consistent with the previous reports suggesting that more than one intracellular loop are involved in the contact with the G protein through mutagenesis studies [13,[15][16][18][19]38]. The putative pocket is further supported by the presence of the key residues, which are important to the G protein coupling identified by mutagenesis, including R60 in the iLP1 [31], R130 and F138 in the iLP2 [38,20], and C223 in the iLP3.…”

Section: Putative G Protein-binding Pocket Formed By the Three Intracsupporting

confidence: 92%

“…Recombinant protein studies have implied that the N-and C-terminal portions of the third intracellular loop (iLP3), and the cytoplasmic tail of the receptors are involved in the G protein activation and signal transduction. Other studies have shown that in the second intracellular loop (iLP2), the membrane-proximal portions of the iLP3, and the N-terminal segment of the cytoplasmic tail both contain amino acids which have been predicted to play a role in regulating the selectivity of G protein and GPCR interactions [13][14][15][16][17][18][19][20][21][22][23].…”

Section: Introductionmentioning

confidence: 99%

Assembling NMR structures for the intracellular loops of the human thromboxane A2 receptor: Implication of the G protein-coupling pocket

Feng

Ruan

2008

Archives of Biochemistry and Biophysics

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It has been reported that the multiple intracellular loops (iLPs) of the thromboxane A 2 receptor (TP) are involved in the receptor G protein coupling. In this study, a high-resolution 2D NMR technique was used to determine the 3D structures of the first, second, and third iLPs of the TP receptor using synthetic peptides constrained into the loop structures. 2D 1H NMR spectra, TOCSY and NOESY were obtained for the two peptides from proton NMR experiments. The NMR data was processed and assigned through the Felix 2000 program. Standard methods were used to acquire sequencespecific assignments. Structure calculations were processed through DGII and NMR refinement programs within the Insight II program. We were able to calculate and use the NOE constraints to obtain the superimposed structure of ten structures for each iLP peptide. The NMR-determined structures of the iLP peptides were used to refine a homology model of the TP receptor. A 3D Gprotein binding cavity, formed by the three intracellular loops, was predicted by the docking of the C-terminal domain of the Gαq. Based on the structural model and the previous mutagenesis, the residues in the TP intracellular loops (R130, R60, C223, F138) and in the Gαq (L360, V361, E358, Y359), which are important for interaction of TP with the G protein, were further highlighted. These results reveal the possibly important molecular mechanisms in TP receptor signaling and provide structural information to characterize other prostanoid receptor signalings.

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Expression of serotonin receptors and role of serotonin in human prostate cancer tissue and cell lines

et al. 2004

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This is the first study demonstrating an overexpression of 5-HTR subtypes 1A and 1B in PC cells, especially in high-grade tumors. Moreover, 5-HT stimulates proliferation of PC cells and 5-HTR1A antagonists inhibit proliferation. Thus, we propose that 5-HT has an important role in tumor progression, especially in the androgen-independent state of the disease. The design of specific antagonists for this type of receptor might be useful for the growth control of androgen-independent tumors.

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Site‐Directed Mutations in the Third Intracellular Loop of the Serotonin 5‐HT_1A Receptor Alter G Protein Coupling from G_i to G_s in a Ligand‐Dependent Manner

Cited by 54 publications

References 20 publications

Molecular Determinants in the Second Intracellular Loop of the 5-Hydroxytryptamine-1A Receptor for G-Protein Coupling

Molecular Determinants in the Second Intracellular Loop of the 5-Hydroxytryptamine-1A Receptor for G-Protein Coupling

Assembling NMR structures for the intracellular loops of the human thromboxane A2 receptor: Implication of the G protein-coupling pocket

Expression of serotonin receptors and role of serotonin in human prostate cancer tissue and cell lines

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