The Second Intracellular Loop of the m5 Muscarinic Receptor Is the Switch Which Enables G-protein Coupling

Burstein, Ethan S.; Spalding, Tracy A.; Brann, Mark R.

doi:10.1074/jbc.273.38.24322

Cited by 119 publications

(128 citation statements)

References 61 publications

Supporting

Mentioning

117

Contrasting

Unclassified

Order By: Relevance

“…A recent study using the m5 muscarinic receptor indicates that the second intracellular loop is also important for enabling G protein activation (Burstein et al, 1998). These studies show that G protein-coupling selectivity is in large part detexmined by the second intracellular loop.…”

Section: G Protein-coupiing Of the Gpcrs And The Dopamine D4 Receptormentioning

confidence: 78%

SH3 Binding Domains in the Dopamine D4 Receptor

Oldenhof

Vickery²,

Anafi³

et al. 1998

Biochemistry

View full text Add to dashboard Cite

The dopamine D4 receptor is a G protein-coupled receptor (GPCR) that belongs to the dopamine D2-like receptor family. Functionally, the D2-like receptors are characterized by their ability to inhibit adenylyl cyclase. The dopamine D4 receptor as well as many other catecholaminergic receptors contain several putative SH3 binding domains. Most of these sites in the D4 receptor are located in a polymorphic repeat sequence and flanking sequences in the third intracellular loop. Here we demonstrate that this region of the D4 receptor can interact with a large variety of SH3 domains of different origin. The strongest interactions were seen with the SH2−SH3 adapter proteins Grb2 and Nck. The repeat sequence itself is not essential in this interaction. The data presented indicate that the different SH3 domains in the adapter proteins interact in a cooperative fashion with two distinct sites immediately upstream and downstream from the repeat sequence. Removal of all the putative SH3 binding domains in the third intracellular loop of the dopamine D4 receptor resulted in a receptor that could still bind spiperone and dopamine. Dopamine could not modulate the coupling of these mutant receptors to adenylyl cyclase and MAPK, although dopamine modulated receptor−G protein interaction appeared normal. The receptor deletion mutants show strong constitutive internalization that may account for the deficiency in functional activation of second messengers. The data indicates that the D4 receptor contains SH3 binding sites and that these sites fall within a region involved in the control of receptor internalization.

show abstract

Section: G Protein-coupiing Of the Gpcrs And The Dopamine D4 Receptormentioning

confidence: 78%

SH3 Binding Domains in the Dopamine D4 Receptor

Oldenhof

Vickery²,

Anafi³

et al. 1998

Biochemistry

View full text Add to dashboard Cite

show abstract

“…Interestingly, when the sequence of the ␣ 1b -AR is aligned with those of the muscarinic M1, M3, and M5 receptors a homologous arginine is similarly located. Mutation of this arginine in the M5 muscarinic receptor to either Asp or Glu also produced constitutive activity (26).…”

Section: Mutagenesis Of Basic Residues In the I2 Loop And Cytosolic Ementioning

confidence: 99%

“…Altogether the features of the V147E mutant are similar to those of the previously described R143E mutant that displayed high affinity for epinephrine, despite being completely impaired in its signaling properties (6). Mutations of the homologous valine in other GPCRs has also resulted in a profound impairment of receptor-G protein coupling (26,28), whereas increased constitutive activity induced by its mutation has not been reported to date.…”

Section: Mutagenesis Of Conserved Hydrophobic Residues Of the ␣ 1b -Amentioning

confidence: 99%

Mutational and Computational Analysis of the α1b-Adrenergic Receptor

Greasley¹,

Fanelli²,

Scheer³

et al. 2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

To investigate their role in receptor coupling to G q , we mutated all basic amino acids and some conserved hydrophobic residues of the cytosolic surface of the ␣ 1b -adrenergic receptor (AR) GPCRs are structurally characterized by seven transmembrane ␣-helices connected by alternating extracellular (e) and intracellular (i) loops. While the extracellular portion of these receptors is primarily involved in ligand binding, the cytosolic loops mediate the interaction of the receptors with a number of signaling and regulatory proteins, including G proteins, arrestins, and G protein-coupled receptor kinases (reviewed in Ref..2).Evidence suggests that a conformational adjustment within the helical bundle of the receptor underlies the process of agonist-induced activation of GPCRs (reviewed in Ref.3). The current hypothesis is that the transition from the inactive (R) to active (R*) state of a GPCR results in receptor interaction with, and activation of, a G protein. Thus a GPCR-mediated biological response involves a series of events (i.e. receptor activation, receptor-G protein interaction, and receptor-induced G protein activation) for which a detailed mechanism still remains elusive at the molecular level. Although residues located in the helical bundle and at the boundary between the membrane and the cytosol may play a role in the "conformational switch" underlying receptor activation, amino acids in the intracellular loops are believed to be more directly involved in receptor-G protein interaction and/or receptor-induced G protein activation. The combination of these two latter events, which cannot be unequivocally separated experimentally, is generally indicated with the term of receptor-G protein coupling.We have previously provided evidence that the negatively and positively charged amino acids of the conserved DRY motif at the cytosolic end of helix 3 play a key role in the activation process of the ␣ 1b -AR (4 -6). Following a combination of experimental and computer-simulated mutagenesis of the ␣ 1b -AR, we have hypothesized that protonation of the aspartate (Asp 142 ) and a shift of the arginine (Arg 143 ) out of a conserved "polar pocket" are crucial steps in the transition of the receptor from the inactive (R) to active (R*) state (4 -6).Several studies have tried to identify the amino acids of different GPCRs involved in G protein coupling at both experimental (as reviewed in Ref.2) and theoretical levels (7-10). The majority of these studies indicate that sequences in the i2 loop as well as in the N and C termini of the i3 loop play an

show abstract

“…Another consequence of RNA editing may be to alter the specificity of activation of heterotrimeric G-proteins and thereby shift intracellular signaling pathways. To test this hypothesis, we examined the ability of three 5-HT 2C R isoforms to functionally couple with the ␣ subunits of various heterotrimeric G-proteins of the G q family (G q , G 11 , G 14 , G 15 , and G 16 ) and the G 12 family (G 12 and G 13 ) using the cell-based functional assay, Receptor Selection/Amplification Technology (R-SAT).…”

mentioning

confidence: 99%

RNA Editing of the Human Serotonin 5-HT2CReceptor Alters Receptor-mediated Activation of G13Protein

Price¹,

Weiner²,

Chang³

et al. 2001

Journal of Biological Chemistry

100

View full text Add to dashboard Cite

The recent completion of the human genome predicted the presence of only 30,000 genes, stressing the importance of mechanisms that increase molecular diversity at the post-transcriptional level. One such posttranscriptional event is RNA editing, which generates multiple protein isoforms from a single gene, often with profound functional consequences. The human serotonin 5-HT 2C receptor undergoes RNA editing that creates multiple receptor isoforms. One consequence of RNA editing of cell surface receptors may be to alter the pattern of activation of heterotrimeric G-proteins and thereby shift preferred intracellular signaling pathways. We examined the ability of the nonedited 5-HT 2C receptor isoform (INI) and two extensively edited isoforms, VSV and VGV, to interact with various G-protein ␣ subunits. Two functional assays were utilized: the cellbased functional assay, Receptor Selection/Amplification Technology TM , in which the pharmacological consequences of co-expression of 5HT 2C receptor isoforms with G-protein ␣ subunits in fibroblasts were studied, and 5HT 2C receptor-mediated rearrangements of the actin cytoskeleton in stable cell lines. These studies revealed that the nonedited 5-HT 2C receptor functionally couples to G q and G 13 . In contrast, coupling to G 13 was not detected for the extensively edited 5-HT 2C receptors. Thus, RNA editing represents a novel mechanism for regulating the pattern of activation of heterotrimeric G-proteins, molecular switches that control an enormous variety of biological processes.The monoamine 5-hydroxytryptamine (serotonin; 5-HT) 1 interacts with a large family of receptors to induce signal transduction events important in the modulation of neurotransmission (1). The 2C subtype of serotonin receptor (5-HT 2C R) is a member of the G-protein-coupled receptor superfamily and interacts with G q to stimulate phospholipase C, resulting in the production of inositol phosphates and diacylglycerol (2). RNA transcripts encoding the human 5-HT 2C R undergo adenosineto-inosine RNA editing events at five positions, termed A, B, C, D, and E (Fig. 1A), altering the amino acid coding potential within the putative second intracellular loop of the protein (3, 4). We and others have demonstrated a decrease in agonist potency at the human edited VSV and VGV isoforms (named for the amino acids at positions 156, 158, and 160) compared with the nonedited isoform, which codes for INI at these positions. This decrease in agonist potency is reflected as a rightward shift in the dose-response curve for inositol phosphate accumulation (5, 6) and calcium release (7). The decreased agonist potency was proposed to result from a reduced G qprotein coupling efficiency induced by the introduction of these novel amino acids into the second intracellular loop, a region known to be important for G-protein coupling (8 -16). Another consequence of RNA editing may be to alter the specificity of activation of heterotrimeric G-proteins and thereby shift intracellular signaling pathways. To test this hypothesi...

show abstract

The Second Intracellular Loop of the m5 Muscarinic Receptor Is the Switch Which Enables G-protein Coupling

Cited by 119 publications

References 61 publications

SH3 Binding Domains in the Dopamine D4 Receptor

SH3 Binding Domains in the Dopamine D4 Receptor

Mutational and Computational Analysis of the α1b-Adrenergic Receptor

RNA Editing of the Human Serotonin 5-HT2CReceptor Alters Receptor-mediated Activation of G13Protein

Contact Info

Product

Resources

About