Madan M. Kwatra

β2-Adrenergic and Several Other G Protein–Coupled Receptors in Human Atrial Membranes Activate Both Gs and Gi

Cardiac G protein–coupled receptors that couple to G&agr;s and stimulate cAMP formation (eg, &bgr;-adrenergic, histamine, serotonin, and glucagon receptors) play a key role in cardiac inotropy. Recent studies in rodent cardiac myocytes and transfected cells have revealed that one of these receptors, the &bgr;2-adrenergic receptor (AR), also couples to the inhibitory G protein G&agr;i (activation of which inhibits cAMP formation). If &bgr;2ARs could be shown to couple to G&agr;i in the human heart, it would have important ramifications, because levels of G&agr;i increase with age and in failing human heart. Therefore, we investigated whether &bgr;2ARs in the human heart activate G&agr;i. By photoaffinity labeling human atrial membranes with [32P]azidoanilido-GTP, followed by immunoprecipitation with antibodies specific for G&agr;i, we found that G&agr;i is activated by stimulation of &bgr;2ARs but not of &bgr;1ARs. In addition, we found that other G&agr;s-coupled receptors also couple to G&agr;i, including histamine, serotonin, and glucagon. When coupling of these receptors to G&agr;i is disrupted by pertussis toxin, their ability to stimulate adenylyl cyclase is enhanced. These data provide the first evidence that &bgr;2AR and many other G&agr;s-coupled receptors in human atrium also couple to G&agr;i and that abolishing the coupling of these receptors to G&agr;i increases the receptor-mediated adenylyl cyclase activity.

A constitutively active form of neurokinin 1 receptor and neurokinin 1 receptor-mediated apoptosis in glioblastomas

Previous studies have shown that neurokinin 1 receptor (NK1R) occurs naturally in human glioblastomas and its stimulation causes cell proliferation. In the present study we show that stimulation of NK1R in human U373 glioblastoma cells by substance P increases Akt phosphorylation by 2.5‐fold, with an EC50 of 57 nM. Blockade of NK1R lowers basal phosphorylation of Akt, indicating the presence of a constitutively active form of NK1R; similar results are seen in U251 MG and DBTRG‐05 glioblastoma cells. Linkage of NK1R to Akt implicates NK1R in apoptosis of glioblastoma cells. Indeed, treatment of serum‐starved U373 cells with substance P reduces apoptosis by 53 ± 1% (p < 0.05), and treatment with NK1R antagonist L‐733,060 increases apoptosis by 64 ± 16% (p < 0.01). Further, the blockade of NK1R in human glioblastoma cells with L‐733,060 causes cleavage of Caspase‐3 and proteolysis of poly (ADP‐ribose) polymerase. Experiments designed to elucidate the mechanism of NK1R‐mediated Akt phosphorylation revealed total involvement of non‐receptor tyrosine kinase Src and phosphatidyl‐3‐kinase, a partial involvement of epidermal growth factor receptor, and no involvement of mitogen‐activated protein/extracellular signal‐related kinase. Taken together, the results of the present study indicate a key role for NK1R in glioblastoma apoptosis.

Human substance P receptor expressed in Chinese hamster ovary cells directly activates Gαq/11, Gαs, and Gαo

Substance P receptor (SPR) stably expressed in Chinese hamster ovary (CHO) cells stimulates at least three second messenger systems including phosphoinositide hydrolysis, cyclic AMP (cAMP) formation, and arachidonic acid release. Whether these second messenger systems are activated via single or multiple G proteins is not known. Therefore, in the present study we examined whether human SPR (hSPR) stably expressed in CHO cells activates multiple G proteins. This was achieved by photoaffinity labeling of Gα‐subunits with [32P]azidoanilido‐GTP ([32P]AA‐GTP) upon hSPR stimulation in CHO‐hSPR membranes followed by immunoprecipitation of the labeled Gα‐subunits with antibodies specific for various Gα‐subunits. These experiments reveal that hSPR directly activates Gαq/11, Gαs and Gαo. While hSPR is known to couple Gαq/11, the present study provides the first evidence that hSPR can also activate Gαs and Gαo in a mammalian system.

Human substance P receptor lacking the C‐terminal domain remains competent to desensitize and internalize

Substance P receptor (SPR) and its naturally occurring splice‐variant, lacking the C‐terminal tail, are found in brain and spinal cord. Whether C‐terminally truncated SPR desensitizes like full‐length SPR is controversial. We used a multivaried approach to determine whether human SPR (hSPR) and a C‐terminally truncated mutant, hSPRΔ325, differ in their desensitization and internalization. In HEK‐293 cells expressing either hSPRΔ325 or hSPR, SP‐induced desensitization of the two receptors was similar when measured by inositol triphosphate accumulation or by transient translocation of coexpressed PKCβII‐GFP to the plasma membrane. Moreover, translocation of β‐arrestin 1 or 2‐GFP (βarr1‐GFP or βarr2‐GFP) to the plasma membrane, and receptor internalization were also similar. However, hSPR and hSPRΔ325 differ in their phosphorylation and in their ability to form β‐arrestin‐containing endocytic vesicles. Unlike hSPR, hSPRΔ325 is not phosphorylated to a detectable level in intact HEK293 cells, and whereas hSPR forms vesicles containing either βarr1‐GFP or βarr2‐GFP, hSPRΔ325 does not form any vesicles with βarr1‐GFP, and forms fewer vesicles with βarr2‐GFP. We conclude that truncated hSPR undergoes agonist‐dependent desensitization and internalization without detectable receptor phosphorylation.

Acute depression of myocardial β-adrenergic receptor signaling during cardiopulmonary bypass. Impairment of the adenylyl cyclase moiety

BACKGROUND Previously the authors showed that myocardial beta-adrenergic (betaAR) function is reduced after cardiopulmonary bypass (CPB) in a canine model Whether CPB results in similar effects on betaAR function in adult humans is not known. Therefore the current study tested two hypotheses: (1) That myocardial betaAR signaling is reduced in adult humans after CPB, and (2) that administration of long-term preoperative betaAR antagonists prevents this process. METHODS After they gave informed consent, 52 patients undergoing aortocoronary surgery were enrolled. Atrial biopsies were obtained before CPB and immediately before discontinuation of CPB. Plasma catecholamine concentrations, myocardial betaAR density, and functional responsiveness (basal, isoproterenol, zinterol, sodium fluoride, and manganese-stimulated adenylyl cyclase activity) were assessed. RESULTS Catecholamine levels increased significantly during CPB (P < 0.005). Myocardial betaAR adenylyl cyclase coupling decreased during CPB, as evidenced by a 21% decrease in isoproterenol-stimulated adenylyl cyclase activity (750 [430] pmol cyclic adenosine monophosphate per milligram total protein 15 min before CPB compared with 540 [390] at the end of CPB, P = 0.0062, medians [interquartile range]) despite constant betaAR density. Differential activation along the betaAR signal transduction cascade localized the defect to the adenylyl cyclase moiety. Administration of long-term preoperative betaAR antagonists did not prevent acute CPB-induced myocardial betaAR dysfunction. CONCLUSIONS These data indicate that the myocardial adenylyl cyclase response to betaAR agonists decreases acutely in adults during aortocoronary surgery requiring CPB, regardless of whether long-term preoperative betaAR antagonists are administered. The mechanism underlying acute betaAR dysfunction appears to be direct impairment of the adenylyl cyclase moiety. Similar increases in manganese-stimulated activity before and at the end of CPB show preserved adenylyl cyclase catalytic activity, suggesting that other mechanisms (such as decreased protein levels or altered isoform expression or function) may be responsible for decreased adenylyl cyclase function.

Transcriptional Regulation of the Human α1a-Adrenergic Receptor Gene CHARACTERIZATION OF THE 5′-REGULATORY AND PROMOTER REGION

We recently cloned cDNAs encoding three subtypes of human α1-adrenergic receptors (α1ARs), α1a, α1b, and α1d (Schwinn, D. A., Johnston, G. L., Page, S. O., Mosley, M. J., Wilson, K. H., Worman, N. P., Campbell, S., Fidock, M. D., Furness, L. M., Parry-Smith, D. J., Peter, B., and Bailey, D. S. (1995) J. Pharmacol. Exp. Ther. 272, 134–142) and demonstrated predominance of α1aARs in many human tissues (Price, D. T., Lefkowitz, R. J., Caron, M. G., Berkowitz, D., and Schwinn, D. A. (1994) Mol. Pharmacol. 45, 171–175). Several lines of evidence indicate that α1aARs are important in clinical diseases such as myocardial hypertrophy and benign prostatic hyperplasia. Therefore, we initiated studies to understand mechanisms underlying regulation of α1aAR gene transcription. A genomic clone containing 6.2 kb of 5′-untranslated region of the human α1aAR gene was recently isolated. Ribonuclease protection and primer extension assays indicate that α1aAR gene transcription occurs at multiple initiation sites with the major site located 696 base pairs upstream of the ATG, where a classic initiator sequence is located. Transfection of luciferase reporter constructs containing varying amounts of 5′-untranslated region into human SK-N-MC neuroblastoma cells indicate that a region extending 125 base pairs upstream from the main transcription initiation site contains full α1aAR promoter activity. Furthermore, distinct activator and suppressor elements lie 2–3 and 3–5 kilobase pairs upstream, respectively. Although the α1aAR promoter contains neither TATA or CAAT elements, gel shift mobility assays targeting three GC boxes immediately upstream of the main transcription initiation site confirm binding of Sp1. Activity of the α1aAR promoter is cell-specific, demonstrating highest activity in cells endogenously expressing α1aARs. The human α1aAR gene also contains several cis regulatory elements, including several insulin and cAMP response elements. Consistent with these observations, we provide the first evidence that treatment of SK-N-MC cells with insulin and cAMP elevating agents leads to an increase in α1aAR expression. In conclusion, these data represent the first characterization of the α1aAR gene; our findings should facilitate further studies designed to understand mechanisms regulating α1AR subtype-specific expression in healthy and diseased human tissue.

Signal transduction through substance P receptor in human glioblastoma cells: roles for Src and PKCδ

Substance P receptor (SPR), a G protein-coupled receptor (GPCR), is found in human glioblastomas, and has been implicated in their growth. Consistent with a role for SPR in cell growth, activation of SPR in U373 MG human glioblastoma cells leads to the phosphorylation of mitogen-activated protein kinases [extracellular signal-regulated kinase 1 and 2 (ERK1/2)] and stimulation of cell proliferation. The purpose of the present study was to elucidate the pathway through which these actions occur. Using either the epidermal growth factor receptor (EGFR) kinase inhibitor, AG 1478, or a small-interfering RNA (siRNA) directed against human EGFR, we found that transactivation of EGFR by SPR is only marginally involved in SP-dependent ERK1/2 phosphorylation. Src, however, is shown to be a major component of SPR signaling because the Src kinase inhibitor, PP2, and a kinase-dead Src mutant both inhibit SP-dependent ERK1/2 phosphorylation. We also report that SPR stimulates the phosphorylation of protein kinase Cδ(PKCδ), and that this stimulation is blocked by PP2. SP-dependent ERK1/2 phosphorylation is also blocked by rottlerin, a PKCδ inhibitor, and the calcium scavenger, BAPTA/AM. Finally, rottlerin and PP2 were both found to inhibit the growth of several glioblastoma cell lines, underscoring the potential of these agents to block glioblastoma growth.

The ligand binding domain of the epidermal growth factor receptor is not required for receptor dimerization

To examine the role of the ligand binding domain of epidermal growth factor receptor in its dimerization, we studied the dimerization of a truncated form of the receptor that resembles v-erbB in that it lacks a ligand binding domain. Receptor dimerization was determined by sedimentation analysis on sucrose density gradients at different concentrations of Triton X-100. At high concentrations of Triton X-100 (0.2%), the truncated receptor occurred as a monomer and displayed low basal autophosphorylation. By contrast, at low concentrations of Triton X-100 (0.01%), it existed as a dimer and exhibited high basal autophosphorylation. The ability of the truncated receptor to dimerize indicates that the ligand binding domain of the epidermal growth factor receptor is not required for receptor dimerization.

Specific photoaffinity labelling of inhibitory adenosine receptors

N6(L-phenylisopropyl)adenosine (L-PIA) and N6(3-iodo-4-azido benzyl)-adenosine (IAzBA) inhibit the adenylate cyclase activity in synaptic membranes of chick cerebellum via Ri adenosine receptors. [3H]L-PIA and [125I]AzBA bind to these membranes with Kd values of approximately 1 nM and Bmax values of approximately 1000 fmol/mg protein. Photolysis of [125I]AzBA bound to synaptic membranes results in the specific incorporation of radioactivity into a protein with Mr = 36,000. This photoincorporation is blocked by simultaneous exposure to L-PIA, theophylline, an adenosine receptor antagonist, or Gpp(NH)p, but not by cytosine, suggesting that the 36,000 dalton protein is the Ri adenosine receptor or a subunit of the receptor that contains the adenosine binding site.

α1,-Adrenoceptor Subtypes in the Human Cardiovascular and Urogenital Systems

Publisher Summary α 1 ARs are important in many human diseases, with the α 1a AR subtype playing a particularly important role in urinary symptoms associated with benign prostatic hypertrophy (BPH). Characterization of prostate α 1 ARs, including tissue-specific regulation, should facilitate the understanding of mechanisms underlying this common disease of elderly men. To study regulation of α 1 ARs in human tissues, human homologues for each α 1 AR subtype are cloned, stably expressed each in rat-1 cells, and their pharmacology is characterized in detail. In addition to subtype selectivity for various ligands, it is found that all three human α 1 AR subtypes couple to phosphoinositide hydrolysis in a pertussis toxin-insensitive manner. To determine which α 1 AR subtypes are likely to be important in specific human diseases, the distribution of each subtype is characterized in various tissues. α 1 AR subtype tissue distribution is unique and varies with the species studied. For example, while the α 1b is present in rat liver, only the a 1a subtype is expressed in native human liver. In addition to subtype distribution, understanding regulation of α 1 ARs (both at an mRNA and protein level) is important in elucidating mechanisms underlying various human diseases. To understand what controls expression of α 1a ARs in BPH. Human cell lines not expressing α 1a ARs endogenously exhibit low transcriptional activity, suggesting cell-specific expression of α 1a ARs. Elucidating mechanisms underlying prostate-specific modulation of α 1a ARs is the goal of present studies. To understand the function and regulation of prostate α 1a ARs at a protein level, studies are initiated to biochemically characterize these receptors. In such studies, as photoaffinity labeling has been claimed to be different between α 1 AR subtypes (absent in the α 1d ), first this biochemical property is examined using cloned α 1 ARs.