A small sequence in the third intracellular loop of the VPAC(1) receptor is responsible for its efficient coupling to the calcium effector

The stimulatory effect of vasoactive intestinal peptide (VIP) on the intracellular calcium concentration ([Ca(2+)](i)) has been investigated in Chinese hamster ovary cells stably transfected with the reporter gene aequorin, and expressing human VPAC(1), VPAC(2), chimaeric VPAC(1)/VPAC(2) or mutated receptors. The VIP-induced increase in [Ca(2+)](i) was linearly correlated with receptor density, and was higher in cells expressing VPAC(1) receptors than in cells expressing a similar density of VPAC(2) receptors. The study was performed to establish the receptor sequence responsible for this difference. VPAC(1)/VPAC(2) chimaeric receptors were first used for broad positioning: those receptors having the third intracellular loop (IC3) of the VPAC(1) or the VPAC(2) receptor behaved, in this respect, phenotypically like VPAC(1) and VPAC(2) receptors respectively. Replacement in the VPAC(2) receptor of the sequence comprising residues 315-318 (VGGN) within IC3 by its VPAC(1) receptor counterpart (residues 328-331; IRKS) and the introduction of VGGN instead of IRKS into VPAC(1) was sufficient to mimic VPAC(1) and VPAC(2) receptor characteristics respectively. Thus a small sequence in the IC3 domain of the VPAC(1) receptor is responsible for the efficient agonist-stimulated increase in [Ca(2+)](i).     

Different domains in the third intracellular loop of the GLP-1 receptor are responsible for Galpha(s) and Galpha(i)/Galpha(o) activation

It has previously been shown that the GLP-1 receptor is primarily coupled to the adenylate cyclase pathway via activation of Galpha(s) proteins. Recent studies have shown that the third intracellular loop of the receptor is important in the stimulation of cAMP production. We have studied the effect of three synthetic peptide sequences derived from the third intracellular loop of the GLP-1 receptor on signal transduction in Rin m5F cell membranes. The whole third intracellular loop strongly stimulates both pertussis toxin and cholera toxin-sensitive G proteins, while the N-terminal half exclusively stimulates cholera toxin-sensitive G proteins and the C-terminal half only stimulates pertussis toxin-sensitive G-proteins as demonstrated by measurements of GTPase activity. These data confirm that the principal stimulatory G-protein interaction site resides in the third intracellular loop, but also suggest that the GLP-1 receptor is not only coupled to the Galpha(s) but also to the Galpha(i)/Galpha(o) type of G proteins and that distinct domains within the third intracellular loop are responsible for the activation of the different G-protein subfamilies.     

Structure and function of the third intracellular loop of the 5-hydroxytryptamine2A receptor: the third intracellular loop is alpha-helical and binds purified arrestins

Understanding the precise structure and function of the intracellular domains of G protein-coupled receptors is essential for understanding how receptors are regulated, and how they transduce their signals from the extracellular milieu to intracellular sites. To understand better the structure and function of the intracellular domain of the 5-hydroxytryptamine2A (5-HT2A) receptor, a model G(alpha)q-coupled receptor, we overexpressed and purified to homogeneity the entire third intracellular loop (i3) of the 5-HT2A receptor, a region previously implicated in G-protein coupling. Circular dichroism spectroscopy of the purified i3 protein was consistent with alpha-helical and beta-loop, -turn, and -sheet structure. Using random peptide phage libraries, we identified several arrestin-like sequences as i3-interacting peptides. We subsequently found that all three known arrestins (beta-arrestin, arrestin-3, and visual arrestin) bound specifically to fusion proteins encoding the i3 loop of the 5-HT(2A) receptor. Competition binding studies with synthetic and recombinant peptides showed that the middle portion of the i3 loop, and not the extreme N and C termini, was likely to be involved in i3-arrestin interactions. Dual-label immunofluorescence confocal microscopic studies of rat cortex indicated that many cortical pyramidal neurons coexpressed arrestins (beta-arrestin or arrestin-3) and 5-HT2A receptors, particularly in intracellular vesicles. Our results demonstrate (a) that the i3 loop of the 5-HT2A receptor represents a structurally ordered domain composed of alpha-helical and beta-loop, -turn, and -sheet regions, (b) that this loop interacts with arrestins in vitro, and is hence active, and (c) that arrestins are colocalized with 5-HT2A receptors in vivo.     

Structure of the third intracellular loop of the human cannabinoid 1 receptor

The third cytoplasmic loop (IC3) is a determinant in the dynamic life cycle of G protein-coupled receptors, including the activation, internalization, desensitization, and resensitization processes. Here, we characterize the structural features of the IC3 of the cannabinoid 1 receptor (CB1) in micelle solution using heteronuclear, (1)H,(15)N-high-resolution NMR methods. The IC3 construct was designed to contain one-third of each of the transmembrane helices (TMs 5 and 6) to tether the protein to the hydrophobic portion of the micelle. Indeed, the NMR analysis illustrates prominent alpha-helices at the N-terminus (G1-R10) and C-terminus (Q37-T47) of the IC3 receptor domain, corresponding to the cytoplasmic termini of TM5 and TM6. The structural features of the central portion of the IC3 consist of a small alpha-helix, adjacent to the terminus of TM5. The remainder is mostly unstructured as indicated by the NMR-based observables (NOEs and chemical shifts). Despite the lack of secondary structure, the hydrophobic triplet of isoleucine residues in the center of the IC3 is found in molecular dynamics simulations to associate with the lipid environment, producing two smaller loops out of the IC3. Previous studies examining mastoparan and related peptides and their ability to activate G proteins have concluded an alpha-helix is required for efficient binding and activation. Our structural results for the IC3 of CB1 would then suggest that in the intact receptor the G protein is activated by the alpha-helices of the cytoplasmic ends of TM5 or TM6 and not the unstructured central region of the IC3.     

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

The effect of mutations (V344E and T343A/V344E) in the third intracellular loop of the serotonin 5-HT(1A) receptor expressed transiently in human embryonic kidney 293 cells have been examined in terms of receptor/G protein interaction and signaling. Serotonin, (R)-8-hydroxy-2-dipropylaminotetralin [(R)-8-OH-DPAT], and buspirone inhibited cyclic AMP production in cells expressing native and mutant 5-HT(1A) receptors. Serotonin, however, produced inverse bell-shaped cyclic AMP concentration-response curves at native and mutant 5-HT(1A) receptors, indicating coupling not only to G(i)/G(o), but also to G(s). (R)-8-OH-DPAT, however, induced stimulation of cyclic AMP production only after inactivation of G(i)/G(o) proteins by pertussis toxin and only at the mutant receptors. The partial agonist buspirone was unable to induce coupling to G(s) at any of the receptors, even after pertussis toxin treatment. The basal activities of native and mutant 5-HT(1A) receptors in suppressing cyclic AMP levels were not found to be significantly different. The receptor binding characteristics of the native and mutant receptors were investigated using the novel 5-HT(1A) receptor antagonist [(3)H]NAD-299. For other receptors, analogous mutations have produced constitutive activation. This does not occur for the 5-HT(1A) receptor, and for this receptor the mutations seem to alter receptor/G protein coupling, allowing ligand-dependent coupling of receptor to G(s) in addition to G(i)/G(o) proteins.     

Arrestin binding to the M(2) muscarinic acetylcholine receptor is precluded by an inhibitory element in the third intracellular loop of the receptor

Desensitization of G protein-coupled receptors (GPCRs) involves the binding of members of the family of arrestins to the receptors. In the model system involving the visual GPCR rhodopsin, activation and phosphorylation of rhodopsin is thought to convert arrestin from a low to high affinity binding state. Phosphorylation of the M(2) muscarinic acetylcholine receptor (mAChR) has been shown to be required for binding of arrestins 2 and 3 in vitro and for arrestin-enhanced internalization in intact cells (Pals-Rylaarsdam, R., and Hosey, M. M. (1997) J. Biol. Chem. 272, 14152-14158). For the M(2) mAChR, arrestin binding requires phosphorylation at multiple serine and threonine residues at amino acids 307-311 in the third intracellular (i3) loop. Here, we have investigated the molecular basis for the requirement of receptor phosphorylation for arrestin binding. Constructs of arrestin 2 that can bind to other GPCRs in a phosphorylation-independent manner were unable to interact with a mutant M(2) mAChR in which the Ser/Thr residues at 307-311 were mutated to alanines. However, although phosphorylation-deficient mutants of the M(2) mAChR that lacked 50-157 amino acids from the i3 loop were unable to undergo agonist-dependent internalization when expressed alone in tsA201 cells, co-expression of arrestin 2 or 3 restored agonist-dependent internalization. Furthermore, a deletion of only 15 amino acids (amino acids 304-319) was sufficient to allow for phosphorylation-independent arrestin-receptor interaction. These results indicate that phosphorylation at residues 307-311 does not appear to be required to activate arrestin into a high affinity binding state. Instead, phosphorylation at residues 307-311 appears to facilitate the removal of an inhibitory constraint that precludes receptor-arrestin association in the absence of receptor phosphorylation.     

A conserved arginine in the distal third intracellular loop of the mu-opioid receptor is required for G protein activation

In the present study, the functional significance of the intracellular C-terminal loop of the mu-opioid receptor in activating Gi proteins was determined by constructing a C-terminal deletion mutant mu(C delta 45) receptor, which lacks the carboxyl 45 amino acids. When the truncated mu(C delta 45) receptor was stably expressed in human embryonic kidney (HEK) 293 cells, the efficacy and the potency of [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO), a specific mu-opioid receptor agonist, to inhibit forskolin-stimulated adenylate cyclase activity were not significantly affected. Similar to other G-coupled receptors, the third cytoplasmic loop of the mu-opioid receptor contains conserved basic residues (R276/R277/R280) at the C-terminal segment. Mutating these basic residues to neutral amino acids (L276/M277/L280) greatly impaired the ability of DAMGO to inhibit forskolin-stimulated cyclic AMP formation. Replacing R276/R277 with L276/M277 did not affect the efficacy and potency by which DAMGO inhibits the adenylate cyclase activity. In HEK 293 cells stably expressing mutant (R280L) mu-opioid receptors, the ability of DAMGO to inhibit forskolin-stimulated cyclic AMP production was greatly reduced. These results suggest that the intracellular carboxyl tail of the mu-opioid receptor does not play a significant role in activating Gi proteins and that the arginine residue (R280) at the distal third cytoplasmic loop is required for Gi activation by the mu-opioid receptor.     

Asn229 in the third helix of VPAC1 receptor is essential for receptor activation but not for receptor phosphorylation and internalization: comparison with Asn216 in VPAC2 receptor

After stimulation with agonist, G protein coupled receptors (GPCR) undergo conformational changes that allow activation of G proteins to transduce the signal, followed by phosphorylation by kinases and arrestin binding to promote receptor internalization. Actual paradigm, based on a study of GPCR-A/rhodopsin family, suggests that a network of interactions between conserved residues located in transmembrane (TM) domains (mainly TM3, TM6 and TM7) is involved in the molecular switch leading to GPCR activation.

We evaluated in CHO cells expressing the VPAC₁ receptor the role of the third transmembrane helix in agonist signalling by point mutation into Ala of the residues highly conserved in the secretin-family of receptors: Y²²⁴, N²²⁹, F²³⁰, W²³², E²³⁶, G²³⁷, Y²³⁹, L²⁴⁰. N²²⁹A VPAC₁ mutant was characterized by a decrease in both potency and efficacy of VIP stimulated adenylate cyclase activity, by the absence of agonist stimulated [Ca²⁺]_i increase, by a preserved receptor recognition of agonists and antagonist and by a preserved sensitivity to GTP suggesting the importance of that residue for efficient G protein activation. N²²⁹D mutant was not expressed at the membrane, and the N²²⁹Q with a conserved mutation was less affected than the A mutant. Agonist stimulated phosphorylation and internalization of N²²⁹A and N²²⁹Q VPAC₁ were unaffected. However, the re-expression of internalized mutant receptors, but not that of the wild type receptor, was rapidly reversed after VIP washing. Receptor phosphorylation, internalization and re-expression may be thus dissociated from G protein activation and linked to another active conformation that may influence its trafficking.

Mutation of that conserved amino acid in VPAC₂ could be investigated only by a conservative mutation (N²¹⁶Q) and led to a receptor with a low VIP stimulation of adenylate cyclase, receptor phosphorylation and internalization. This indicated the importance of the conserved N residue in the TM3 of that family of receptors.     

Proline residue 280 in the second extracellular loop (EC2) of the VPAC2 receptor is essential for the receptor structure

Inspection of the amino acid sequence of the human VPAC1 and the VPAC2 receptors after alignment of the conserved residues indicates that the second extracellular loop (EC2) is one amino acid shorter in the VPAC1 receptor due to the lack of a proline residue in position 294. We hypothesized that this could be of importance for receptor structure and/or for ligand recognition. Insertion by directed mutagenesis of a proline in that position (294 VPAC1) had little consequence on the binding of several agonists but reduced the affinity for the VPAC1 antagonist. Coupling of the 294 VPAC1 receptor to adenylate cyclase was improved, as demonstrated by an increased affinity for VIP and other agonists, and by a shift of the VPAC1 antagonist to partial agonist behavior. Deletion of the proline 280 (DeltaPro280 VPAC2) in the VPAC2 receptor markedly reduced the apparent affinity for all the agonists tested. Replacement of the proline by a glycine residue had a smaller effect on the ligands affinities. The proline residue in the VPAC2 receptor EC2 is thus essential for the receptor structure, and the EC2 domain is involved in ligand recognition and receptor functionality.     

The II-III loop of the skeletal muscle dihydropyridine receptor is responsible for the Bi-directional coupling with the ryanodine receptor.

The dihydropyridine receptor (DHPR) in the skeletal muscle plasmalemma functions as both voltage-gated Ca(2+) channel and voltage sensor for excitation-contraction (EC) coupling. As voltage sensor, the DHPR regulates intracellular Ca(2+) release via the skeletal isoform of the ryanodine receptor (RyR-1). Interaction with RyR-1 also feeds back to increase the Ca(2+) current mediated by the DHPR. To identify regions of the DHPR important for receiving this signal from RyR-1, we expressed in dysgenic myotubes a chimera (SkLC) having skeletal (Sk) DHPR sequence except for a cardiac (C) II-III loop (L). Tagging with green fluorescent protein (GFP) enabled identification of expressing myotubes. Dysgenic myotubes expressing GFP-SkLC or SkLC lacked EC coupling and had very small Ca(2+) currents. Introducing a short skeletal segment (alpha(1S) residues 720-765) into the cardiac II-III loop (replacing alpha(1C) residues 851-896) of GFP-SkLC restored both EC coupling and Ca(2+) current densities like those of the wild type skeletal DHPR. This 46-amino acid stretch of skeletal sequence was recently shown to be capable of transferring strong, skeletal-type EC coupling to an otherwise cardiac DHPR (Nakai, J., Tanabe, T., Konno, T., Adams, B., and Beam, K.G. (1998) J. Biol. Chem. 273, 24983-24986). Thus, this segment of the skeletal II-III loop contains a motif required for both skeletal-type EC coupling and RyR-1-mediated enhancement of Ca(2+) current.     

The third intracellular loop stabilizes the inactive state of the neuropeptide Y1 receptor

Constitutively active G-protein-coupled receptors (GPCRs) can signal even in the absence of ligand binding. Most Class I GPCRs are stabilized in the resting conformation by intramolecular interactions involving transmembrane domain (TM) 3 and TM6, particularly at loci 6.30 and 6.34 of TM6. Signaling by Gi/Go-coupled receptors such as the Neuropeptide Y1 receptor decreases already low basal metabolite levels. Thus, we examined constitutive activity using a biochemical assay mediated by a Gi/Gq chimeric protein and a more direct electrophysiological assay. Wild-type (WT-Y1) receptors express no measurable, agonist-independent activation, while mu-opioid receptors (MOR) and P2Y12 purinoceptors showed clear evidence of constitutive activation, especially in the electrophysiological assay. Neither point mutations at TM6 (T6.30A or N6.34A) nor substitution of the entire TM3 and TM6 regions from the MOR into the Y1 receptor increased basal WT-Y1 activation. By contrast, chimeric substitution of the third intracellular loop (ICL3) generated a constitutively active, Y1-ICL3-MOR chimera. Furthermore, the loss of stabilizing interactions from the native ICL3 enhanced the role of surrounding residues to permit basal receptor activation; because constitutive activity of the Y1-ICL3-MOR chimera was further increased by point mutation at locus 6.34, which did not alter WT-Y1 receptor activity. Our results indicate that the ICL3 stabilizes the Y1 receptor in the inactive state and confers structural properties critical for regulating Y receptor activation and signal transduction. These studies reveal the active participation of the ICL3 in the stabilization and activation of Class I GPCRs.     

Lysine 270 in the third intracellular domain of the oxytocin receptor is an important determinant for G alpha(q) coupling specificity

To identify structural elements important to specific G alpha(q) coupling in the oxytocin receptor (OTR), intracellular domains were exchanged between OTR and G alpha(s)-coupled vasopressin V(2) receptors (V(2)Rs). Substitution of sequence from the second (2i) and third (3i) intracellular domains of V(2)R into comparable positions in OTR markedly reduced ligand affinity and resulted in a loss of G alpha(q) coupling. Substitution of the 2i domain of OTR into V(2)R decreased ligand affinity and vasopressin-stimulated adenylyl cyclase activity and only slightly increased phosphatidylinositide turnover. In contrast, substitution of the OTR3i domain into V(2)R produced a receptor chimera with high ligand affinity, decreased vasopressin-stimulated adenylyl cyclase activity, and markedly enhanced ligand-stimulated phosphatidylinositide turnover. The C-terminal 36 amino acids, but not the N-terminal 13 amino acids, of the OTR3i domain contained the determinants critical for enhanced activation of PLC. Mutation of a single lysine in the C-terminal OTR3i sequence to the corresponding V(2)R residue (valine) eliminated the enhanced ability of the V(2)R chimera to stimulate PLC but did not affect maximal adenylyl cyclase stimulation. Furthermore, mutation of this residue (K270) in wild-type OTR completely abolished the ability of the receptor to stimulate phosphatidylinositide turnover, with only a small reduction in ligand affinity. These data demonstrate that OTR K270 is critically important in the stimulation by OTR of phosphatidylinositide turnover and that this determinant can also increase this activity in the V(2)R chimera. Mutation of K270 also adversely affects the ability of OTR to stimulate ERK1/2 phosphorylation. Therefore, this residue plays an important role in the specificity of OTR/G alpha(q)/PLC coupling.     

Phenylalanine 138 in the second intracellular loop of human thromboxane receptor is critical for receptor-G-protein coupling.

Eicosanoid receptors exhibit a highly conserved ERY(C)XXV(I)XXPL sequence in the second intracellular loop. The carboxyl end of this motif contains a bulky hydrophobic amino acid (L,I,V, or F). In human thromboxane A2 receptor (TXA(2)R), phenylalanine 138 is located at the carboxyl end of this highly conserved motif. This study examined the function of the F138 in G protein coupling. F138 was mutated to aspartic acid (D) and tyrosine (Y), respectively. Both mutants F138D and F138Y showed similar ligand binding activity to that of the wild type TXA(2)R. The Kd and Bmax values of either mutant were comparable to those of the wild type receptor. However, both mutants showed significant impairment of agonist induced Ca(2+) signaling and phospholipase C activation. These results suggest that the F138 plays a key role in G protein coupling.     

NMR structure of an intracellular third loop peptide of human GABA(B) receptor

GABA_B receptor is a G protein-coupled receptor for GABA and drug target for neurological and psychiatric disorders. From the analysis of GTPγS binding assay, we found that a synthesized peptide (GABAb: ETKSVSTEKINDHR) corresponding to the intracellular third loop region of metabotropic GABA_B receptor could activate G_i protein α subunit directly. The three dimensional molecular structure of the peptide in SDS-d₂₅ micelles was determined by 2D ¹H-NMR spectroscopy. GABAb peptide formed an α helical structure and a positive charge cluster at the C-terminal site. These structural features were also found in several other G protein activating peptides. From the comparison among these peptides, we found that peptides with high helical content show the high activity.     

Regulation of melanin-concentrating hormone receptor 1 signaling by RGS8 with the receptor third intracellular loop

Melanin-concentrating hormone (MCH) receptor 1 (MCH1R) belongs to the class A G protein-coupled receptors (GPCRs). The MCH-MCH1R system plays a central role in energy metabolism, and thus the regulation of signaling pathways activated by this receptor is of particular interest. Regulator of G protein signaling (RGS) proteins work by increasing the GTPase activity of G protein alpha subunits and attenuate cellular responses coupled with G proteins. Recent evidence has shown that RGS proteins are not simple G protein regulators but equally inhibit the signaling from various GPCRs. Here, we demonstrate that RGS8, which is highly expressed in the brain, functions as a negative modulator of MCH1R signaling. By using biochemical approaches, RGS8 was found to selectively and directly bind to the third intracellular (i3) loop of MCH1R in vitro. When expressed in HEK293T cells, RGS8 and MCH1R colocalized to the plasma membrane and RGS8 potently inhibited the calcium mobilization induced by MCH. The N-terminal 9 amino acids of RGS8 were required for the optimal capacity to downregulate the receptor signaling. Furthermore, Arg(253) and Arg(256) at the distal end of the i3 loop were found to comprise a structurally important site for the functional interaction with RGS8, since coexpression of RGS8 with R253Q/R256Q mutant receptors resulted in a loss of induction of MCH-stimulated calcium mobilization. This functional association suggests that RGS8 may represent a new therapeutic target for the development of novel pharmaceutical agents.     

A region of the third intracellular loop of the short form of the D2 dopamine receptor dictates Gi coupling specificity

The D2 dopamine receptor has two isoforms, the short form (D2s receptor) and the long form (D2l receptor), which differ by the presence of a 29-amino acid insert in the third cytoplasmic loop. Both the D2s and D2l receptors have been shown to couple to members of the G alpha(i) family of G proteins, but whether each isoform couples to specific G alpha(i) protein(s) remains controversial. In previous studies using G alpha(i) mutants resistant to modification by pertussis toxin (G alpha(i)PT), we demonstrated that the D2s receptor couples selectively to G alpha(i2)PT and that the D2l receptor couples selectively to G alpha(i3)PT (Senogles, S. E. (1994) J. Biol. Chem. 269, 23120-23127). In this study, two point mutations of the D2s receptor were created by random mutagenesis (R233G and A234T). The two mutant D2s receptors demonstrated pharmacological characteristics comparable with those of the wild-type D2s receptor, with similar agonist and antagonist binding affinities. We used human embryonic kidney 293 cells stably transfected with G alpha(i1)PT, G alpha(i2)PT, or G alpha(i3)PT to measure agonist-mediated inhibition of forskolin-stimulated cAMP accumulation before and after pertussis toxin treatment. The two mutant D2s receptors demonstrated a change in G(i) coupling specificity compared with the wild-type D2s receptor. Whereas the wild-type D2s receptor coupled predominantly to G alpha(i2)PT, mutant R233G coupled preferentially to G alpha(i3)PT, and mutant A234T coupled preferentially to G alpha(i1)PT. These results suggest that this region of the third cytoplasmic loop is crucial for determining G(i) protein coupling specificity.     

A four amino acid deletion polymorphism in the third intracellular loop of the human alpha 2C-adrenergic receptor confers impaired coupling to multiple effectors

The alpha(2)-adrenergic receptors (alpha(2)ARs) play a critical role in modulating neurotransmitter release in the central and peripheral sympathetic nervous systems. A polymorphism of the alpha(2)AR subtype localized to human chromosome 4 (the pharmacologic alpha(2C)AR subtype) within an intracellular domain has been identified in normal individuals. The polymorphism (denoted Del322-325) is because of an in-frame 12-nucleic acid deletion encoding a receptor lacking Gly-Ala-Gly-Pro in the third intracellular loop. To delineate the functional consequences of this structural alteration, Chinese hamster ovary cells were permanently transfected with constructs encoding wild-type human alpha(2C)AR and the polymorphic receptor. The Del322-325 variant had decreased high affinity agonist binding (K(H) = 7.3 +/- 0.95 versus 3.7 +/- 0.43 nm; %R(H) = 31 +/- 4 versus 49 +/- 4) compared with wild-type indicating impaired formation of the agonist-receptor-G protein complex. The polymorphic receptor displayed markedly depressed epinephrine-promoted coupling to G(i), inhibiting adenylyl cyclase by 10 +/- 4.3% compared with 73 +/- 2.4% for wild-type alpha(2C)AR. This also was so for the endogenous ligand norepinephrine and full and partial synthetic agonists. Depressed agonist-promoted coupling to the stimulation of MAP kinase ( approximately 71% impaired) and inositol phosphate production ( approximately 60% impaired) was also found with the polymorphic receptor. The Del322-325 receptor was approximately 10 times more frequent in African-Americans compared with Caucasians (allele frequencies 0.381 versus 0.040). Given this significant loss of function phenotype in several signal transduction cascades and the skewed ethnic prevalence, Del322-325 represents a pharmacoethnogenetic locus and may also be the basis for interindividual variation in cardiovascular or central nervous system pathophysiology.     

An asn to lys polymorphism in the third intracellular loop of the human alpha 2A-adrenergic receptor imparts enhanced agonist-promoted Gi coupling

alpha(2A)-Adrenergic receptors (alpha(2A)AR) are presynaptic autoinhibitory receptors of noradrenergic neurons in the central and peripheral sympathetic nervous systems, which act to dynamically regulate neurotransmitter release. Signaling through the G(i)/G(o) family of G-proteins, the receptor subserves numerous homeostatic and central nervous system functions. A single nucleotide polymorphism of this receptor, which results in an Asn to Lys substitution at amino acid 251 of the third intracellular loop, was identified in the human population. The frequency of Lys-251 was 10-fold greater in African-Americans than in Caucasians, but was not associated with essential hypertension. To determine the consequences of this substitution, wild-type and Lys-251 receptors were expressed in CHO and COS-7 cells. Expression, ligand binding, and basal receptor function were unaffected by the substitution. However, agonist-promoted [(35)S]GTPgammaS binding was approximately 40% greater with the Lys-251 receptor. This enhanced agonist function was observed with catecholamines, azepines, and imidazolines albeit to different degrees. In studies of agonist-promoted functional coupling to G(i), the polymorphic receptor displayed enhanced inhibition of adenylyl cyclase (60 +/- 4. 4 versus 46 +/- 4.1% inhibition) and markedly enhanced stimulation of MAP kinase (57 +/- 9 versus 15- +/- 2-fold increase over basal) compared with wild-type alpha(2A)AR. The potency of epinephrine in stimulating inositol phosphate accumulation was increased approximately 4 fold with the Lys-251 receptor. Unlike previously described variants of G-protein-coupled receptors, where the minor species causes either a loss of function or increased non-agonist function, Lys-251 alpha(2A)AR represents a new class of polymorphism whose phenotype is a gain of agonist-promoted function.     

Mutations in the carboxy-terminus of the third intracellular loop of the human recombinant VPAC1 receptor impair VIP-stimulated [Ca2+]i increase but not adenylate cyclase stimulation

The vasoactive intestinal polypeptide (VIP) VPAC1 receptor is preferentially coupled to Galphas protein that stimulates adenylate cyclase activity and also to Galphaq and Galphai proteins that stimulate the inositol phosphate/calcium pathway. Previous studies indicated the importance of the third intracellular loop of the receptor for G protein coupling. By site-directed mutation of the human recombinant receptor expressed in Chinese hamster ovary cells, we identified two domains in this loop that contain clusters of basic residues conserved in most of the G-protein-coupled seven transmembrane domains receptors. We found that mutations in the proximal domain (K322) reduced the capability of VIP to increase adenylate cyclase activity without any change in the calcium response, whereas mutations in the distal part of the loop (R338, L339, R341) markedly reduced the calcium increase and Galphai coupling but only weakly the adenylate cyclase activity. Thus, the interaction of different G proteins with the VPAC1 receptor involves different receptor sub-domains.     

Identification of G-proteins coupling to the vasoactive intestinal peptide receptor VPAC(1) using immunoaffinity chromatography: evidence for precoupling.

VPAC(1) receptor subtype-specific G-protein interactions were identified using a strategy that exploits an essential initial signaling event, namely the functional and physical association of the receptor with G-protein. An immunoaffinity purification column was constructed using a previously characterized antibody that had been raised against the first extracellular loop of the VPAC(1) receptor. VPAC(1)/G-protein complexes were solubilized from membranes and copurified. Receptor and Galpha-proteins were detected in eluates using (125)I-VIP labeling and immunoblotting, respectively. Human VPAC(1) transfected in HEK293 cells couples to Gs but not Gi3, Gi1/2, or Gq. Rat VPAC(1) in brain membranes is coupled to Gs and Gi3. Rat VPAC(1) in lung membranes couples to Gs, Gi3, and Gq. Pretreatment of membranes with VIP increased the level of all G-proteins copurifying with VPAC(1). Immunoaffinity chromatography also revealed VPAC(1) receptor precoupling to G-protein in the absence of VIP pretreatment. This was confirmed using a cross-linking procedure to capture VIP receptor/G-protein complexes in the native membrane milieu prior to solubilization. Precoupling suggests that there is a significant basal level of VPAC(1) receptor activity especially in cells, such as some human malignant tumor cells, that express high levels of receptor.