Modulation Of Receptor Function Research Articles

G-protein-coupled receptor dimers in the lime light

It will be important to apply these optical resonance energy transfer techniques to examine the dimerization of other GPCRs in intact cells. GPCRs can be classified into three major subfamilies (rhodopsin, calcitonin and metabotropic receptor subfamilies) and members of all three subfamilies have been shown to exist as homodimers12xSee all References12. Several recent studies have also revealed heterodimerization of GPCRs (between closely related as well as distantly related members), which alters the functional properties of these receptors13xGABAB receptors – the first 7TM heterodimers. Marshall, F.H. et al. Trends Pharmacol. Sci. 1999; 20: 396–399Abstract | Full Text | Full Text PDF | PubMed | Scopus (245)See all References, 14xG-protein-coupled receptor hetero-dimerization modulates receptor function. Jordan, B.A. and Devi, L.A. Nature. 1999; 399: 697–700Crossref | PubMed | Scopus (833)See all References, 15xReceptors for dopamine and somatostatin: formation of hetero-oligomers with enhanced functional activity. Rocheville, M. et al. Science. 2000; 288: 154–157Crossref | PubMed | Scopus (638)See all References. Biochemical analyses have shown that in some GPCRs, agonist treatment leads to stabilization and/or an increase in the level of dimers, whereas in others agonist treatment has no effect or leads to a decrease in the level of dimers12xSee all References12. These optical resonance energy transfer techniques provide a novel and clear protocol to examine both homodimerization and heterodimerization of GPCRs in living cells. These methods are free from the complications that arise from more traditional biochemical methods of identifying dimers, and we should expect to see more studies using BRET and FRET given their simplicity and straightforwardness.Dimerization of GPCRs is a novel mechanism for receptor activation. It remains to be established whether GPCR dimerization occurs in vivo (i.e. in a living organism). Recent studies using energy transfer techniques to demonstrate the existence of dimers in living cells is a major step in this direction.

Theoretical study on mutation-induced activation of the luteinizing hormone receptor

Here, three-dimensional model building and molecular dynamics simulations of the luteinizing hormone receptor have been employed to generate hypotheses about the molecular mechanisms underlying the activation of the receptor induced by naturally occurring activating mutations. The comparative analysis of the wild-type receptor and of 16 constitutively active or inactive mutants has been instrumental in inferring the structural/dynamic features which could characterize the inactive and the active forms of the receptor. These features have been also employed for predicting the functional behavior of new receptor mutants.The results of this study might provide a structural framework to interpret the pathological effects induced by mutations of the luteinizing hormone receptor. In addition, the proposed theoretical model could be useful for engineering new mutations or ligands able to modulate receptor function.

Kappa opioid receptor endocytosis by dynorphin peptides.

Internalization and downregulation are important steps in the modulation of receptor function. Recent work with the beta2 adrenergic and opioid receptors have implicated these processes in receptor-mediated activation of mitogen-activated protein kinase (MAPK). We have used CHO cells expressing epitope-tagged rat kappa opioid receptors (rKORs) and prodynorphin-derived peptides to characterize the agonist-mediated endocytosis of rKORs and activation of MAPK. Kappa receptor-selective peptides induced receptor internalization and downregulation whereas nonpeptide agonists did not. An examination of the ability of dynorphin A-17-related peptides (lacking C-terminal amino acids) to promote KOR internalization, inhibition of adenylyl cyclase, and MAPK phosphorylation revealed that the N-terminal seven residues play an important role in eliciting these responses. Both dynorphin peptides and nonpeptide agonists induced rapid and robust phosphorylation of MAPKs. Taken together, these results point to a difference in the ability of dynorphin peptides and nonpeptide ligands to promote rKOR endocytosis and support the view that rKOR internalization is not required for MAPK activation.

Nuclear run-on assays for measurement of adrenergic receptor transcription rate.

The functionally diverse group of G-protein-coupled receptors (GPCRs) is a superfamily of membrane receptors. They include receptors for many different signaling molecules, such as peptide and nonpeptide hormones, neurotransmitters, chemokines, prostanoids, and proteinases. The principal function of GPCRs is to transmit information about the extracellular environment to the interior of the cell by interacting with the heterotrimeric G-proteins and, thereby, participate in regulation of many cellular functions. In view of their major importance, it is not surprising that GPCR-mediated responses are subject to dynamic regulation by a number of mechanisms. These regulatory mechanisms have important roles in fine-tuning signals from multiple receptor signaling pathways. Multiple mechanisms contribute to the regulation of GPCRs and their transmembrane signaling. Posttranslational modifications of the receptors, such as phosphorylation, may modulate receptor function; in addition, changes in receptor gene expression can lead to alterations in sensitivity and responsiveness of cells to various signaling molecules (, , ).

C-Src-mediated Phosphorylation of the Epidermal Growth Factor Receptor on Tyr845 and Tyr1101 Is Associated with Modulation of Receptor Function

Accumulating evidence indicates that interactions between the epidermal growth factor receptor (EGFR) and the nonreceptor tyrosine kinase c-Src may contribute to an aggressive phenotype in multiple human tumors. Previous work from our laboratory demonstrated that murine fibroblasts which overexpress both these tyrosine kinases display synergistic increases in DNA synthesis, soft agar growth, and tumor formation in nude mice, and increased phosphorylation of the receptor substrates Shc and phospholipase gamma as compared with single overexpressors. These parameters correlated with the ability of c-Src and EGFR to form an EGF-dependent heterocomplex in vivo. Here we provide evidence that association between c-Src and EGFR can occur directly, as shown by receptor overlay experiments, and that it results in the appearance of two novel tyrosine phosphorylations on the receptor that are seen both in vitro and in vivo following EGF stimulation. Edman degradation analyses and co-migration of synthetic peptides with EGFR-derived tryptic phosphopeptides identify these sites as Tyr845 and Tyr1101. Tyr1101 lies within the carboxyl-terminal region of the EGFR among sites of receptor autophosphorylation, while Tyr845 resides in the catalytic domain, in a position analogous to Tyr416 of c-Src. Phosphorylation of Tyr416 and homologous residues in other tyrosine kinase receptors has been shown to be required for or to increase catalytic activity, suggesting that c-Src can influence EGFR activity by mediating phosphorylation of Tyr845. Indeed, EGF-induced phosphorylation of Tyr845 was increased in MDA468 human breast cancer cells engineered to overexpress c-Src as compared with parental MDA 468 cells. Furthermore, transient expression of a Y845F variant EGFR in murine fibroblasts resulted in an ablation of EGF-induced DNA synthesis to nonstimulated levels. Together, these data support the hypothesis that c-Src-mediated phosphorylation of EGFR Tyr845 is involved in regulation of receptor function, as well as in tumor progression.

Modulation of GABAA receptor function by neuroactive steroids: evidence for heterogeneity of steroid sensitivity of recombinant GABAA receptor isoforms

Neuroactive steroids are potent, selective allosteric modulators of gamma-aminobutyric acid type A (GABAA) receptor function in the central nervous system, and may serve as endogenous anxiolytic and analgesic agents. In order to study the influence of subunit subtypes of the GABAA receptor on modulation of receptor function by neuroactive steroids, we expressed human recombinant GABAA receptors in Xenopus oocytes. GABA-activated membrane current, and the modulatory effects of the endogenous neurosteroid 5alpha-pregnan-3alpha-ol-20-one (allopregnanolone) and the synthetic steroid anesthetic 5alpha-pregnan-3alpha-ol-11,20-dione (alphaxalone) were measured using two-electrode voltage-clamp recording techniques. Allopregnanolone had similar effects to potentiate GABA-activated membrane current in the alpha1beta1gamma2L and alpha1beta2gamma2L receptor isoforms. In contrast, alphaxalone was much more effective as a positive allosteric modulator on the alpha1beta1gamma2L receptor isoform. In the absence of the gamma2L subunit subtype, allopregnanolone had much greater efficacy, but its potency was decreased. Allopregnanolone was much more effective on the alpha1beta1 receptor isoform compared with the alpha1beta2 receptor isoform. The potency for alphaxalone to potentiate the GABA response was not altered in the absence of the gamma2L subunit subtype, although its efficacy was greatly enhanced. Both allopregnanolone and alphaxalone produced nonparallel leftward shifts in the GABA concentration-response relationship in the absence of the gamma2L subunit, decreasing the EC50 concentration of GABA and increasing the maximal response. Only alphaxalone increased the maximal GABA response when the gamma2L subunit subtype was present. The 3beta-pregnane isomers epipregnanolone and isopregnanolone both inhibited the ability of allopregnanolone and alphaxalone to potentiate GABAA receptor function. However, the degree of block produced by the 3beta-pregnane steroid isomers was dependent on the type of receptor isoform studied and the neuroactive steroid tested. Isopregnanolone, the 3beta-isomer of allopregnanolone, was significantly more effective as a blocker of potentiation caused by allopregnanolone compared with alphaxalone in all receptor isoforms tested. Epipregnanolone had a greater efficacy as a blocker at the alpha1beta2gamma2L receptor isoform compared with the alpha1beta1gamma2L receptor isoform, and also produced a greater degree of block of potentiation caused by allopregnanolone compared with alphaxalone. Our results support the hypothesis that the heteromeric assembly of different GABAA receptor isoforms containing different subunit subtypes results in multiple steroid recognition sites on GABAA receptors, which in turn produces distinctly different modulatory interactions between neuroactive steroids acting at the GABAA receptor. The alpha and gamma subunit subtypes may have the greatest influence on allopregnanolone modulation of GABAA receptor function, whereas the beta and gamma subunit subtypes appear to be most important for the modulatory effects of alphaxalone.Key words: GABAA receptor, neurosteroid, allopregnanolone, alphaxalone, Xenopus oocyte.

Structure and function of the ovine type 1 corticotropin releasing factor receptor (CRF 1) and a carboxyl-terminal variant

Corticotropin releasing factor (CRF) is the major neuropeptide regulating the hypothalamo–pituitary–adrenocortical axis in most species. A pituitary receptor for CRF (designated CRF 1) belonging to the seven-transmembrane helix, G-protein-coupled receptor superfamily has been cloned for human, rat, mouse and xenopus. Since ovine CRF shares only 84% identity to human/rat CRF (h/rCRF) we postulated that the sheep pituitary CRF 1 receptor may have similarly diverged from the rodent and human CRF 1. We report the molecular cloning of an ovine pituitary cDNA containing a 1245 bp open reading frame encoding a 415 amino acid sheep CRF 1 receptor 78, 86, 94, and 95% homologous to xenopus, chicken, rat, mouse, and human CRF 1, respectively. The divergence in primary structure between the sheep CRF 1 and the other mammalian CRF 1s is primarily localized to the extracellular amino terminal domain of the receptor (18 of 22 divergent residues, ovine vs human CRF 1). A variant of the oCRF 1 was also isolated (oCRF 1var) with 133 bp deleted from nucleotide (nt) 1080 to nt 1213 of the open reading frame (ORF) resulting in a new ORF of 1176 nt predicting a 392 residue CRF 1 variant receptor. The 133 bp deletion would cause a frame-shift at residue 358 within the carboxyl-third of the seventh transmembrane domain (TM7) resulting in a shortened cytoplasmic tail with a new amino acid sequence from residue 358 to 392. Scatchard analysis of saturation curves using membrane prepared from Cos 7 cells transfected with oCRF 1 or oCRF 1var indicated that both wild-type and variant receptors were expressed similarly (number of CRF binding sites) and both bound oCRF with high affinity [oCRF 1 ( K d): 2.5+1.6 nM; oCRF 1var: 5.1+2.3 nM]. The non-hydrolyzable GTP analogue (GTP γS) lowered the affinity of both wild-type and variant oCRF 1 receptors to a similar extent (oCRF 1: 18.2 nM; oCRF 1var: 22.4 nM). Both wild-type and variant oCRF 1 receptors exhibited ≈10-fold greater selectivity for oCRF and sauvagine compared to h/rCRF or α-helical [9–41]oCRF. CRF effectively stimulated the accumulation of cAMP (EC 50=51 pM) in Cos 7 cells transiently transfected with wild-type but not variant oCRF 1 receptor. In Cos 7 cells transfected with oCRF 1var, cAMP accumulation was only observed at the highest concentration of oCRF utilized (100 nM). Basal (unstimulated) levels of cAMP in Cos 7 cells transfected with oCRF 1var (in the presence of 2 mM IBMX) were ≈50% lower than for the wild-type oCRF 1. Differences in cAMP accumulation could not be attributed to differences in receptor number since total binding sites in the transfected cells were not different between wild-type or variant oCRF 1 receptors. Agonist-induced receptor internalization, determined as the percent of total [ 125I] Tyr 0-oCRF bound located in the acid-resistant fraction of transfected Cos 7 cells, increased with time (0–60 min at 37°C) for both wild-type and variant oCRF 1. Wild-type CRF 1 internalized ≈2-fold greater percent of total [ 125I] Tyr 0-oCRF bound compared to the variant receptor. In summary, an ovine CRF 1 and a CRF 1 cytoplasmic tail receptor variant displaying high affinity binding to oCRF as well as selectivity for oCRF vs h/rCRF, were cloned from an adult sheep pituitary cDNA library. GTP γS studies indicate that both variant and wild-type receptors couple efficiently to G αs however, only the wild-type oCRF 1 is capable of stimulating cAMP production at physiological levels of CRF. Agonist-induced internalization of the ovine CRF 1var is also reduced compared to the wild-type CRF 1 receptor. We suggest that the oCRF 1var interacts efficiently with G αs but is unable (post-hormonal binding) to effectively stimulate G-protein activation of adenylate cyclase, indicating that the cytoplasmic tail of the CRF 1 can modulate receptor function related to signal transduction. The cytoplasmic carboxyl terminus of the CRF 1 also appears to play a role in agonist-induced internalization of this member of the class II subfamily of G-protein coupled receptors (GPCRs).

Interaction of the N-Ethylmaleimide–Sensitive Factor with AMPA Receptors

Glutamate receptors mediate the majority of rapid excitatory synaptic transmission in the central nervous system (CNS) and play important roles in synaptic plasticity and neuronal development. Recently, protein–protein interactions with the C-terminal domain of glutamate receptor subunits have been shown to be involved in the modulation of receptor function and clustering at excitatory synapses. In this paper, we have found that the N-ethylmaleimide–sensitive factor (NSF), a protein involved in membrane fusion events, specifically interacts with the C terminus of the GluR2 and GluR4c subunits of AMPA receptors in vitro and in vivo. Moreover, intracellular perfusion of neurons with a synthetic peptide that competes with the interaction of NSF and AMPA receptor subunits rapidly decreases the amplitude of miniature excitatory postsynaptic currents (mEPSCs), suggesting that NSF regulates AMPA receptor function.

Modulation of GABA(A) receptor function by neuroactive steroids: evidence for heterogeneity of steroid sensitivity of recombinant GABA(A) receptor isoforms.

Neuroactive steroids are potent, selective allosteric modulators of gamma-aminobutyric acid type A (GABA(A)) receptor function in the central nervous system, and may serve as endogenous anxiolytic and analgesic agents. In order to study the influence of subunit subtypes of the GABA(A) receptor on modulation of receptor function by neuroactive steroids, we expressed human recombinant GABA(A) receptors in Xenopus oocytes. GABA-activated membrane current, and the modulatory effects of the endogenous neurosteroid 5alpha-pregnan-3alpha-ol-20-one (allopregnanolone) and the synthetic steroid anesthetic 5alpha-pregnan-3alpha-ol-11,20-dione (alphaxalone) were measured using two-electrode voltage-clamp recording techniques. Allopregnanolone had similar effects to potentiate GABA-activated membrane current in the alpha1beta1gamma2L and alpha1beta2gamma2L receptor isoforms. In contrast, alphaxalone was much more effective as a positive allosteric modulator on the alpha1beta1gamma2L receptor isoform. In the absence of the gamma2L subunit subtype, allopregnanolone had much greater efficacy, but its potency was decreased. Allopregnanolone was much more effective on the alpha1beta1 receptor isoform compared with the alpha1beta2 receptor isoform. The potency for alphaxalone to potentiate the GABA response was not altered in the absence of the gamma2L subunit subtype, although its efficacy was greatly enhanced. Both allopregnanolone and alphaxalone produced nonparallel leftward shifts in the GABA concentration-response relationship in the absence of the gamma2L subunit, decreasing the EC50 concentration of GABA and increasing the maximal response. Only alphaxalone increased the maximal GABA response when the gamma2L subunit subtype was present. The 3beta-pregnane isomers epipregnanolone and isopregnanolone both inhibited the ability of allopregnanolone and alphaxalone to potentiate GABA(A) receptor function. However, the degree of block produced by the 3beta-pregnane steroid isomers was dependent on the type of receptor isoform studied and the neuroactive steroid tested. Isopregnanolone, the 3beta-isomer of allopregnanolone, was significantly more effective as a blocker of potentiation caused by allopregnanolone compared with alphaxalone in all receptor isoforms tested. Epipregnanolone had a greater efficacy as a blocker at the alpha1beta2gamma2L receptor isoform compared with the alpha1beta1gamma2L receptor isoform, and also produced a greater degree of block of potentiation caused by allopregnanolone compared with alphaxalone. Our results support the hypothesis that the heteromeric assembly of different GABA(A) receptor isoforms containing different subunit subtypes results in multiple steroid recognition sites on GABA(A) receptors, which in turn produces distinctly different modulatory interactions between neuroactive steroids acting at the GABA(A) receptor. The alpha and gamma subunit subtypes may have the greatest influence on allopregnanolone modulation of GABA(A) receptor function, whereas the beta and gamma subunit subtypes appear to be most important for the modulatory effects of alphaxalone.

Ubiquitous molecular substrates for associative learning and activity-dependent neuronal facilitation.

Recent evidence suggests that many of the molecular cascades and substrates that contribute to learning-related forms of neuronal plasticity may be conserved across ostensibly disparate model systems. Notably, the facilitation of neuronal excitability and synaptic transmission that contribute to associative learning in Aplysia and Hermissenda, as well as associative LTP in hippocampal CA1 cells, all require (or are enhanced by) the convergence of a transient elevation in intracellular Ca2+ with transmitter binding to metabotropic cell-surface receptors. This temporal convergence of Ca2+ and G-protein-stimulated second-messenger cascades synergistically stimulates several classes of serine/threonine protein kinases, which in turn modulate receptor function or cell excitability through the phosphorylation of ion channels. We present a summary of the biophysical and molecular constituents of neuronal and synaptic facilitation in each of these three model systems. Although specific components of the underlying molecular cascades differ across these three systems, fundamental aspects of these cascades are widely conserved, leading to the conclusion that the conceptual semblance of these superficially disparate systems is far greater than is generally acknowledged. We suggest that the elucidation of mechanistic similarities between different systems will ultimately fulfill the goal of the model systems approach, that is, the description of critical and ubiquitous features of neuronal and synaptic events that contribute to memory induction.

Cholesterol as Modulator of Receptor Function

The modulatory effect of cholesterol on the function of two structurally related peptide receptors, the oxytocin receptor and the brain cholecystokinin receptor in plasma membranes as well as in intact cells, was analyzed. Different approaches for cholesterol modification were applied: (i) depletion and reloading of cholesterol mediated by methyl-beta-cyclodextrin and cholesterol-methyl-beta-cyclodextrin, respectively, in a reversible manner; (ii) mild treatment of the plasma membranes with cholesterol oxidase under control of the membrane fluidity as measured by fluorescence anisotropy of diphenylhexatriene; and (iii) filipin pretreatment of membranes. The results allowed us to distinguish two mechanisms of cholesterol affecting the ligand-binding function of receptors: changes of the membrane fluidity as demonstrated for the cholecystokinin receptor, or a putatively specific cholesterol-receptor interaction as shown for the oxytocin receptor. This was confirmed in a structure-activity analysis with a variety of sterol analogues substituting for cholesterol in the membranes. While the agonist binding of the cholecystokinin receptor was supported by each of the tested steroids and was well correlated with the corresponding fluorescence anisotropy values, a stringent and unique requirement of the oxytocin receptor's affinity state for structural features of the sterol molecule was found. The molecular requirements differ both from those postulated for sterol-phospholipid interactions and from those known to be necessary for the functional activity of other proteins. The different behavior of both peptide receptors concerning the cholesterol dependence of their ligand binding was also present in vivo at the level of signal transduction. The results suggest that cholesterol can modulate receptor function by two distinct mechanisms, by changes of the membrane fluidity, and/or by a highly specific molecular interaction.

Neurosteroid modulation of [ 3H]flunitrazepam binding in the medulla: an autoradiographic study

Neurosteroids bind to unique sites on the GABA A receptor complex and modulate receptor function. The effects of neurosteroids on GABA A receptors have been well characterized in forebrain regions. However, little is known about their effects on GABA A receptors in the medulla, especially those areas involved in autonomic reflex pathways. Stimulation of [ 3H]flunitrazepam binding to the GABA A receptor by two progesterone metabolites, 3 α-hydroxy-5 α-pregnan-20-one (3 α-OH-DHP) and 3 β-hydroxy-5 α-pregnan-20-one (3 β-OH-DHP), was studied using autoradiographic methods in the medulla and cerebellum of female rats at estrus. [ 3H]Flunitrazepam binding was enhanced by 3 α-OH-DHP in every nucleus examined in the medulla and cerebellum. This effect was stereoselective since 3 β-OH-DHP had no effect on binding in any region. No differences were observed in the degree of stimulation of [ 3H]flunitrazepam binding by 3 α-OH-DHP among medullary brain regions. However, in the cerebellum, the stimulation of binding was significantly greater in the granular layer than in the molecular layer. Stimulation of [ 3H]flunitrazepam binding by 3 α-OH-DHP in nuclei involved in the baroreflex pathways supports previous studies which report that neurosteroids modulate autonomic regulation of blood pressure. These actions may also underlie alterations in autonomic function during pregnancy.

Regulation of angiotensin II receptor AT1 subtypes in renal afferent arterioles during chronic changes in sodium diet.

Studies determined the effects of chronic changes in sodium diet on the expression, regulation, and function of different angiotensin II (ANG II) receptor subtypes in renal resistance vessels. Rats were fed low- or high-sodium diets for 3 wk before study. Receptor function was assessed in vivo by measuring transient renal blood flow responses to bolus injections of ANG II (2 ng) into the renal artery. ANG II produced less pronounced renal vasoconstriction in rats fed a low- compared with high-sodium diet (16% vs. 56% decrease in renal blood flow, P < 0.001). After acute blockade of ANG II formation by iv enalaprilat injection in sodium-restricted animals, ANG II produced a 40% decrease in renal blood flow, a level between untreated dietary groups and less than high salt diet. Intrarenal administration of angiotensin II receptor type 1 (AT1) receptor antagonists losartan or EXP-3174 simultaneously with ANG II caused dose-dependent inhibition of ANG II responses. Based on maximum vasoconstriction normalized to 100% ANG II effect in each group, AT1 receptor antagonists produced the same degree of blockade in all groups, with an apparent maximum of 80-90%. In contrast, similar doses of the angiotensin II receptor type 2 (AT2) receptor ligand CGP-42112 had only a weak inhibitory effect. In vitro equilibrium-saturation 125I-ANG II binding studies on freshly isolated afferent arterioles indicated that ANG II receptor density was lower in the low- vs. high-sodium animals (157 vs. 298 fmol/mg, P < 0.04); affinity was similar (0.65 nM). Losartan and EXP-3174 displaced up to 80-90% of the ANG II binding; fractional displacement was similar in both diet groups. In contrast, the AT2 receptor analogues PD-123319 and CGP-42112 at concentrations < 10(-6) M had no effect on ANG II binding. RT-PCR assays revealed the expression of both angiotensin II receptor type 1A (AT(1A)) and angiotensin II receptor type 1B (AT(1B)) subtypes in freshly isolated afferent arterioles, while there was very little AT2 receptor expression. Total AT1 receptor mRNA expression was suppressed by low sodium intake to 66% of control levels, whereas it was increased to 132% of control by high-sodium diet, as indicated by ribonuclease protection assay. Receptor regulation was associated with parallel changes in AT(1A) and AT(1B) expression; the AT(1A)/AT(1B) ratio was stable at 3.7. We conclude that AT1 receptors are the predominant ANG II receptor type in renal resistance vessels of 7-wk-old rats. Chronic changes in sodium intake caused parallel regulation of expression and amount of receptor protein of the two AT1 receptor genes that modulate receptor function and altered reactivity of renal vessels to ANG II.

Phosphorylation of the Third Intracellular Loop of the Mouse α1b-Adrenergic Receptor by cAMP-dependent Protein Kinase

The third intracellular loop of adrenergic receptors has been implicated in their interaction with guanine nucleotide-binding proteins (G proteins). One of the mechanisms involved in the modulation of receptor function is the phosphorylation of specific residues by intracellular kinases. α1b-Adrenergic receptor is phosphorylated in vitro by cAMP-dependent protein kinase (PKA), although its physiological effect remains to be determined. We have produced fusion proteins formed by glutathione S-transferase and sequences of the third intracellular loop of mouse α1a-, α1b-, and α1d-adrenergic receptor subtypes, and used them as substrates for PKA. Only the fusion protein containing the α1b sequence was phosphorylated in vitro by this kinase. Site-directed mutagenesis of a serine (homologue to serine 278 of the rat sequence, RSS) to an alanine residue precluded phosphorylation by PKA.

Inhibition of the insulin receptor tyrosine kinase by phosphatidic acid.

The lipid second messenger, phosphatidic acid, inhibits the intrinsic tyrosine kinase activity of the insulin receptor in detergent-lipid mixed micelles or in reconstituted membranes. Enzymatic studies revealed that this lipid second messenger inhibits the catalytic activity of partially purified insulin receptor without affecting the affinity of the receptor for insulin. Selectivity in the protein-lipid interaction is suggested by the inability of several other acidic lipids to affect the kinase activity of the receptor and by the relative insensitivity of the inhibition to increasing ionic strength and, in some cases, micelle surface charge. Lysophosphatidic acid and phosphatidic acids with short acyl chains do not affect significantly the receptor's kinase activity, suggesting that hydrophobic interactions are involved in the inhibition. Thus, both a high affinity interaction of the insulin receptor with the phosphate headgroup and a stabilizing hydrophobic interaction with the acyl chains contribute to the inhibitory protein-lipid interaction. The selective sensitivity of the insulin receptor to phosphatidic acid suggests that the receptor-mediated generation of this lipid in the plasma membrane could negatively modulate insulin receptor function.

Aglycone modulation of glycolipid receptor function

The aglycone has been largely ignored in consideration of glycoconjugate function. Evidence is reviewed which suggests that the role of the lipid in glycolipid carbohydrate function may be particularly significant. The lipid moiety can promote or reduce carbohydrate exposure of membrane glycolipids. Theoretical calculation has indicated that the plane of the plasma membrane can restrict the permitted conformations of a given glycolipid oligosaccharide. Thus the lipid moiety may influence the relative conformation of such carbohydrate sequences. Evidence of ceramide regulation of glycolipid function can be found in studies of enzyme substrate specificity, antiglycolipid recognition and bacterial/host cell interactions. Studies of verotoxin binding to its glycolipid receptor globotriaosyl ceramide indicate that modulation of receptor function by glycolipid fatty acid content plays an important role in in vitro binding assays, cell cytotoxicity and intracellular routing.

Tumor-specific expression and alternate splicing of messenger ribonucleic acid encoding activin/transforming growth factor-beta receptors in human pituitary adenomas.

Activin, a member of the transforming growth factor-beta (TGF beta) cytokine family, acts as a pituitary cell mitogen via a novel family of receptor-linked serine/threonine (Ser/Thr) kinases. Pituitary tumors synthesize activin subunits, and the autocrine action of these growth factors may modulate tumor proliferation. We, therefore, investigated the expression of activin/TGF beta type I receptor messenger ribonucleic acids (mRNAs), designated ALK1 through ALK5 (ALK = activin receptor-like kinase), and type II receptor mRNAs using RT-PCR in 34 human pituitary adenomas of all phenotypes and normal pituitary tissue. ALK2 and ALK5, specific mediators of activin and TGF beta signals, respectively, were found to be expressed only in tumor and not in normal pituitary cells, and ALK2 expression was found only in tumors of a mammosomatotroph cell lineage. ALK1, ALK3, and ALK4 mRNAs were found in both normal and neoplastic pituitary cells. The alternatively spliced cytoplasmic domain of ALK4 consists of 11 kinase subdomains, that are critical for modulating receptor function and intracellular signaling. Truncated forms of the ALK4 cytoplasmic domain lacking these subdomains may attenuate activin signal transduction and affect both tumor phenotype and proliferation via the formation of inactive type I/type II complexes. Three truncated ALK4 receptor mRNAs generated by alternate splicing of the cytoplasmic Ser/Thr kinase domain were found to be tumor specific. One of these truncated receptor mRNAs, ALK4-5, is a novel splice variant that has not been previously described. Expression of the ActRII and T beta RII type II receptor mRNAs, which specifically bind activin and TGF beta, respectively, was highly prevalent among all tumor subtypes and normal pituitary tissue. However, ActRIIB, an activin-specific type II receptor that displays a 3- to 4-fold higher affinity for ligand than ActRII, was expressed in 94% of tumors, but was not prevalent in normal tissue. These data are the first to demonstrate tumor-specific expression of Ser/Thr kinase receptors mRNAs and their splice variants in human pituitary adenomas.

Regulation of human interleukin-8 receptor A: identification of a phosphorylation site involved in modulating receptor functions.

The human type A interleukin-8 receptor (IL-8RA) was modified to express an amino-terminal epitope tag and stably overexpressed in a rat basophilic leukemia cell line (RBL-2H3). This receptor (ET-IL-8RA) displayed functional properties similar to those of the native receptor in neutrophils in that exposure to IL-8 stimulated GTPase activity, phosphoinositide (PI) hydrolysis, intracellular calcium mobilization, and degranulation in a pertussis toxin (PTx) susceptible fashion. IL-8 induced dose- and time-dependent phosphorylation of ET-IL-8RA. Phorbol 12-myristate 13-acetate (PMA) treatment also resulted in phosphorylation of the receptor although to a lesser extent. Staurosporine totally blocked PMA-induced phosphorylation but only partially inhibited IL-8-mediated phosphorylation. Phosphorylation of ET-IL-8RA correlated with its desensitization as measured by GTPase activation and calcium mobilization. To determine the role of phosphorylation in IL-8RA signal transduction, three mutants lacking specific serine and threonine residues located at the C-terminal of this receptor were constructed by site-directed mutagenesis (M1, M2, and M3). The mutated receptors expressed in RBL-2H3 cells displayed pharmacological properties (Kd approximately 2-2.8 nM and Bmax approximately 3-3.5 pmol/mg of protein) similar to those of the wild-type ET-IL-8RA. M2 and M3, but not M1, showed a marked decrease in IL-8-induced phosphorylation compared to the wild-type receptor. M2 and M3 but not M1 were resistant to PMA-mediated phosphorylation and desensitization and were also more resistant to homologous desensitization than M1 or ET-IL-8RA.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of ATP on ligand recognition of platelet fibrinogen receptor on GPIIb-IIIa.

The recent discovery of 8-azido-ATP binding sites on the platelet fibrinogen receptor glycoprotein complex GPIIb-IIIa suggests that extracellular ATP may directly modulate function of GPIIb-IIIa. In this study we investigated the effect of ATP on ligand binding to GPIIb-IIIa. Fibrinogen-mediated aggregation of washed platelets was inhibited by ATP and 8-azido-ATP in a dose-dependent manner, independent of the agonist (thrombin, collagen, epinephrine, phorbol 12-myristate 13-acetate) used to induce platelet activation. In addition, 8-azido-ATP and ATP inhibited binding of 125I-labeled fibrinogen to thrombin- and phorbol ester-activated platelets. Interaction of nonstimulated platelets with solid-phase fibrinogen was also reduced by 8-azido-ATP and ATP. Moreover, fibrinogen mimetic peptide-induced conformational change of GPIIb-IIIa on resting platelets was reduced in the presence of both nucleotides. Finally, photoincorporation of 8-azido-[gamma-32P]ATP into GPIIb-IIIa was suppressed by GRGDSP but not by the biologically inactive GRGESP peptide. Thus interaction of ATP with 8-azido-ATP binding sites present on GPIIb-IIIa modulate receptor function, which may play a role in regulation of in vivo platelet aggregation.

Actions of the general anesthetic propofol (2,6‐diisopropylphenol) on the binding of [3h]nicotine to rat cortical membranes

Abstract[3H]nicotine bound to rat cortical membranes in a biphasic manner consistent with previous models of receptor function. The kinetics of binding were characterized by a rapid component with a rate constant of 0.007 min−1 nM−1 and a slower component with an apparent rate constant of 0.024 min−1. Dissociation was monophasic with a rate constant of 0.046 min−1. Addition of 3 mM propofol caused the abolition of the slow component of association and increased the rate constants for the rapid component to 0.012 min−1 and for dissociation to 0.086 min−1. Under non‐equilibrium conditions propofol caused a dose‐dependent increase in the binding of [3]Hnicotine, which reached 147% of the control value at 3 mM propofol. The equilibrium binding of [3H]nicotine was unaffected by the drug. These results are consistent with propofol causing enhanced desensitization at the cortical nicotinic acetylcholine receptor, suggesting an alternative to receptor agonists which bind to active sites as a method of modulating receptor function. © 1994 Wiley‐Liss, Inc.