Mutational analysis of the mouse 5‐HT<sub>7</sub> receptor: importance of the third intracellular loop for receptor–G‐protein interaction

Obosi, Louis A.; Hen, René; Beadle, David J.; Bermúdez, Isabel; King, Linda A.

doi:10.1016/s0014-5793(97)00813-2

Cited by 30 publications

(22 citation statements)

References 17 publications

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“…The insertion of GFP into the third-intracellular loop had no apparent effect on coupling, as SER-7B and SER-7BI3GFP had nearly identical EC 50 s for the 5-HTdependent stimulation of cAMP levels, when the receptors were transiently expressed in COS-7 cells (30 6 3 nm vs. 47 6 5 nm, respectively, Figure 2). In addition, the expression of a modified SER-7B (SER-7B E314A/ K316A I3GFP) designed to couple less effectively to Gproteins by mutation of key amino acids in the thirdintracellular loop of the receptor ( Figure 1B; Obosi et al 1997), also failed to generate robust transgenic lines under the control of the same promoter . As predicted, when SER-7B E314A/K316A I3GFP was expressed in COS-7 cells, the 5-HT stimulation of cAMP levels was dramatically reduced compared to SER-7B, with an EC 50 of 1.8 6 0.5 mM and a maximal activation of less than half that of SER-7B, suggesting that SER-7B E314A/K316 I3GFP was functional, but attenuated (Figure 2).…”

Section: Resultsmentioning

confidence: 99%

SER-7, aCaenorhabditis elegans5-HT7-like Receptor, Is Essential for the 5-HT Stimulation of Pharyngeal Pumping and Egg Laying

et al. 2006

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Serotonin (5-HT) stimulates both pharyngeal pumping and egg laying in Caenorhabditis elegans. Four distinct 5-HT receptors have been partially characterized, but little is known about their function in vivo. SER-7 exhibits most sequence identity to the mammalian 5-HT 7 receptors and couples to a stimulation of adenyl cyclase when expressed in COS-7 cells. However, many 5-HT 7 -specific agonists have low affinity for SER-7. 5-HT fails to stimulate pharyngeal pumping and the firing of the MC motorneurons in animals containing the putative ser-7(tm1325) and ser-7(tm1728) null alleles. In addition, although pumping on bacteria is upregulated in ser-7(tm1325) animals, pumping is more irregular. A similar failure to maintain ''fast pumping'' on bacteria also was observed in ser-1(ok345) and tph-1(mg280) animals that contain putative null alleles of a 5-HT 2 -like receptor and tryptophan hydroxylase, respectively, suggesting that serotonergic signaling, although not essential for the upregulation of pumping on bacteria, ''fine tunes'' the process. 5-HT also fails to stimulate egg laying in ser-7(tm1325), ser-1(ok345), and ser-7(tm1325) ser-1(ok345) animals, but only the ser-7 ser-1 double mutants exhibit an Egl phenotype. All of the SER-7 mutant phenotypes are rescued by the expression of full-length ser-7Tgfp translational fusions. ser-7Tgfp is expressed in several pharyngeal neurons, including the MC, M2, M3, M4, and M5, and in vulval muscle. Interestingly, 5-HT inhibits egg laying and pharyngeal pumping in ser-7 null mutants and the 5-HT inhibition of egg laying, but not pumping, is abolished in ser-7(tm1325);ser-4(ok512) double mutants. Taken together, these results suggest that SER-7 is essential for the 5-HT stimulation of both egg laying and pharyngeal pumping, but that other signaling pathways can probably fulfill similar roles in vivo.

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Section: Resultsmentioning

confidence: 99%

SER-7, aCaenorhabditis elegans5-HT7-like Receptor, Is Essential for the 5-HT Stimulation of Pharyngeal Pumping and Egg Laying

et al. 2006

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“…2002), cytosolic cationic clusters can serve as docking sites for regulatory proteins (Obosi et al . 1997; Wang, , ), and can anchor proteins to negatively charged head groups of phospholipids in the inner leaflet of lipid bilayer (van Klompenburg et al . 1997).…”

Section: Discussionmentioning

confidence: 99%

Exploring the autoinhibitory domain of the electrogenic Na⁺/HCO₃⁻ transporter NBCe1‐B, from residues 28 to 62

Lee

Boron

2018

The Journal of Physiology

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Variant B of the electrogenic Na /HCO cotransporter (NBCe1-B) contributes to the vectorial transport of HCO in epithelia (e.g. pancreatic ducts) and to the maintenance of intracellular pH in the central nervous systems (e.g. astrocytes). NBCe1-B has very low basal activity due to an autoinhibitory domain (AID) located, at least in part, in the unique portion (residues 1-85) of the cytosolic NH -terminus. Previous work has shown that removing 23 amino acids (residues 40-62) stimulates NBCe1-B. Here, we test the hypothesis that a cationic cluster of nine consecutive positively charged amino acids (residues 40-48) is a necessary part of the AID. Using two-electrode voltage clamping of Xenopus oocytes, we assess the activity of human NBCe1-B constructs in which we systematically replace or delete residues 28-62, which includes the cationic cluster. We find that replacing or deleting all residues within the cationic cluster markedly increases NBCe1-B activity (i.e. eliminates autoinhibition). On the background of a cationic clusterless construct, systematically restoring Arg residues restores autoinhibition in two distinct quanta, with one to three Arg residues restoring ∼50%, and four or more Arg residues restoring virtually all autoinhibition. Systematically deleting residues before the cluster reduces autoinhibition by, at most, a small amount. Replacing or deleting residues after the cluster has no effect. For constructs with low NBCe1 activity (but good surface expression, as assessed by biotinylation), co-expression with super-IRBIT (lacking PP1-binding site) restores full activity (i.e. relieves autoinhibition). In summary, the cationic cluster is a necessary component of the AID of NBCe1-B.

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“…Mutation of the basic residues in a motif present at the carboxyl-terminal end of the third intracellular loop of many class A GPCRs, having the sequence BBxxB or BxxBB, has been shown to decrease or eliminate agonist-induced G protein activity (24,36,40,42,47,68). Some studies have demonstrated that exchanging the third intracellular loop regions of distinct GPCRs can result in a shift in the specificity of the interacting G protein (71,72).…”

Section: Discussionmentioning

confidence: 99%

Differential determinants for coupling of distinct G proteins with the class B secretin receptor

Garcia

Dong

Miller

2012

American Journal of Physiology-Cell Physiology

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The secretin receptor is a prototypic class B G protein-coupled receptor that is activated by binding of its natural peptide ligand. The signaling effects of this receptor are mediated by coupling with Gs, which activates cAMP production, and Gq, which activates intracellular calcium mobilization. We have explored the molecular basis for the coupling of each of these G proteins to this receptor using systematic site-directed mutagenesis of key residues within each of the intracellular loop regions, and studying ligand binding and secretin-stimulated cAMP and calcium responses. Mutation of a conserved histidine in the first intracellular loop (H157A and H157R) markedly reduced cell surface expression, resulting in marked reduction in cAMP and elimination of measurable calcium responses. Mutation of an arginine (R153A) in the first intracellular loop reduced calcium, but not cAMP responses. Mutation of a dibasic motif in the second intracellular loop (R231A/K232A) had no significant effects on any measured responses. Mutations in the third intracellular loop involving adjacent lysine and leucine residues (K302A/L303A) or two arginine residues separated by a leucine and an alanine (R318A/R321A) significantly reduced cAMP responses, while the latter also reduced calcium responses. Additive effects were elicited by combining the effective mutations, while combining all the effective mutations resulted in a construct that continued to bind secretin normally, but that elicited no significant cAMP or calcium responses. These data suggest that, while some receptor determinants are clearly shared, there are also distinct determinants for coupling with each of these G proteins.

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Mutational analysis of the mouse 5‐HT₇ receptor: importance of the third intracellular loop for receptor–G‐protein interaction

Cited by 30 publications

References 17 publications

SER-7, aCaenorhabditis elegans5-HT7-like Receptor, Is Essential for the 5-HT Stimulation of Pharyngeal Pumping and Egg Laying

SER-7, aCaenorhabditis elegans5-HT7-like Receptor, Is Essential for the 5-HT Stimulation of Pharyngeal Pumping and Egg Laying

Exploring the autoinhibitory domain of the electrogenic Na⁺/HCO₃⁻ transporter NBCe1‐B, from residues 28 to 62

Differential determinants for coupling of distinct G proteins with the class B secretin receptor

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Mutational analysis of the mouse 5‐HT7 receptor: importance of the third intracellular loop for receptor–G‐protein interaction

Cited by 30 publications

References 17 publications

SER-7, aCaenorhabditis elegans5-HT7-like Receptor, Is Essential for the 5-HT Stimulation of Pharyngeal Pumping and Egg Laying

SER-7, aCaenorhabditis elegans5-HT7-like Receptor, Is Essential for the 5-HT Stimulation of Pharyngeal Pumping and Egg Laying

Exploring the autoinhibitory domain of the electrogenic Na+/HCO3− transporter NBCe1‐B, from residues 28 to 62

Differential determinants for coupling of distinct G proteins with the class B secretin receptor

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Product

Resources

About

Mutational analysis of the mouse 5‐HT₇ receptor: importance of the third intracellular loop for receptor–G‐protein interaction

Exploring the autoinhibitory domain of the electrogenic Na⁺/HCO₃⁻ transporter NBCe1‐B, from residues 28 to 62