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AIMers are short, chemically modified oligonucleotides that induce A-to-I RNA editing through interaction with endogenous adenosine deaminases acting on RNA (ADAR) enzymes. Here, we describe the development of new AIMer designs with base, sugar and backbone modifications that improve RNA editing efficiency over our previous design. AIMers incorporating a novel pattern of backbone and 2' sugar modifications support enhanced editing efficiency across multiple sequences. Further efficiency gains were achieved through incorporation of an N-3-uridine (N3U), in place of cytidine (C), in the 'orphan base' position opposite the edit site. Molecular modeling suggests that N3U might enhance ADAR catalytic activity by stabilizing the AIMer-ADAR interaction and potentially reducing the energy required to flip the target base into the active site. Supporting this hypothesis, AIMers containing N3U consistently enhanced RNA editing over those containing C across multiple target sequences and multiple nearest neighbor sequence combinations. AIMers combining N3U and the novel pattern of 2' sugar chemistry and backbone modifications improved RNA editing both in vitro and in vivo. We provide detailed N3U synthesis methods and, for the first time, explore the impact of N3U and its analogs on ADAR-mediated RNA editing efficiency and targetable sequence space., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Objective: Both microbubble ultrasound contrast agents and acoustic phase change droplets (APCD) have been explored in hepatocellular carcinoma (HCC). This work aimed to evaluate changes to the HCC microenvironment following either microbubble or APCD destruction in a syngeneic pre-clinical model., Methods: Mouse RIL-175 HCC tumors were grown in the right flank of 64 immunocompetent mice. Pre-treatment, photoacoustic volumetric tumor oxygenation, and power Doppler measurements were obtained using a Vevo 3100 system (VisualSonics, Toronto, Canada). The experimental groups received a 0.1 mL bolus injection of either Definity ultrasound contrast agent (Lantheus Medical Imaging) or APCD fabricated by condensing Definity. Following injection, ultrasound destruction was performed using flash-replenishment sequences on a Sequoia with a 10L4 probe (Siemens) for the duration of enhancement. Tumor oxygenation and power Doppler measurements were then repeated immediately post-ultrasound treatment. Twenty-four hours post-treatment, animals were euthanized, and tumors were harvested and stained for CD31, Cleaved Caspase 3 and CD45., Results: Imaging biomarkers demonstrated a significant reduction in percent vascularity following either microbubble or APCD destruction in the tumor microenvironment ( p < 0.022) but no significant changes in tumor oxygenation (p = 0.12). Similarly, immunohistochemistry data demonstrated a significant decrease in CD31 expression (p < 0.042) and an increase in apoptosis (p < 0.014) in tumors treated with destroyed microbubbles or APCD relative to controls. Finally, a significant increase in CD45 expression was observed in tumors treated with APCD (p = 0.046), indicating an increase in tumor immune response., Conclusion: Ultrasound-triggered destruction of both microbubbles and APCD reduces vascularity, increases apoptosis, and may also increase immune response in this HCC model., Competing Interests: Conflict of Interest CEW: Consultant for Bracco Diagnostics, speaking bureau for Canon Medical Systems USA, consultant for SonoSim. JRE: Equipment, drug, grant support and honorarium from GE Healthcare. Drug support from Bracco. Drug, grant support and honorarium from Lantheus Medical Imaging. Equipment support from Siemens. Consultant for SonoSim. Royalties from Elsevier. HF, QL, MAW, JBL: None., (Copyright © 2024 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Huntington's disease (HD) is an autosomal dominant disease caused by the expansion of cytosine-adenine-guanine (CAG) repeats in one copy of the HTT gene (mutant HTT, mHTT). The unaffected HTT gene encodes wild-type HTT (wtHTT) protein, which supports processes important for the health and function of the central nervous system. Selective lowering of mHTT for the treatment of HD may provide a benefit over nonselective HTT-lowering approaches, as it aims to preserve the beneficial activities of wtHTT. Targeting a heterozygous single-nucleotide polymorphism (SNP) where the targeted variant is on the mHTT gene is one strategy for achieving allele-selective activity. Herein, we investigated whether stereopure phosphorothioate (PS)- and phosphoryl guanidine (PN)-containing oligonucleotides can direct allele-selective mHTT lowering by targeting rs362273 (SNP3). We demonstrate that our SNP3-targeting molecules are potent, durable, and selective for mHTT in vitro and in vivo in mouse models. Through comparisons with a surrogate for the nonselective investigational compound tominersen, we also demonstrate that allele-selective molecules display equivalent potency toward mHTT with improved durability while sparing wtHTT. Our preclinical findings support the advancement of WVE-003, an investigational allele-selective compound currently in clinical testing (NCT05032196) for the treatment of patients with HD., Competing Interests: N.I., Y.L., M.F.-K., A.M., W.C.T., K.T., N.K., A.A., K.B., J.D.S., A.L., X.S.H., H.G.J., P.K., F.L., K.L., R.L., M., J.M., Q.P., E.P.-E., M.S., P.S.P., S.S., H.Y., A.Z., C.F., M.B., E.D., and C.V. were employees of Wave Life Sciences during the completion of this work. M. is currently an employee of Stoke Therapeutics. G.L.V. is on the board of directors and is a consultant and shareholder for Wave Life Sciences., (© 2024 The Author(s).)

Background: A suboptimal response to the 2-dose COVID-19 vaccine series in the immunocompromised population prompted recommendations for a 3rd primary dose. We aimed to determine the humoral and cellular immune response to the 3rd COVID-19 vaccine in immunocompromised children., Methods: Prospective cohort study of immunocompromised participants, 5-21 years old, who received 2 prior doses of an mRNA COVID-19 vaccine. Humoral and CD4/CD8 T-cell responses were measured to SARS-CoV-2 spike antigens prior to receiving the 3rd vaccine dose and 3-4 weeks after the 3rd dose was given., Results: Of the 37 participants, approximately half were solid organ transplant recipients. The majority (86.5%) had a detectable humoral response after the 2nd and 3rd vaccine doses, with a significant increase in antibody levels after the 3rd dose. Positive T-cell responses increased from being present in 86.5% to 100% of the cohort after the 3rd dose., Conclusions: Most immunocompromised children mount a humoral and cellular immune response to the 2-dose COVID-19 vaccine series, which is significantly augmented after receiving the 3rd vaccine dose. This supports the utility of the 3rd vaccine dose and the rationale for ongoing emphasis for vaccination against COVID-19 in this population., Impact: Most immunocompromised children mount a humoral and cellular immune response to the 2-dose COVID-19 vaccine series, which is significantly augmented after receiving the 3rd vaccine dose. This is the first prospective cohort study to analyze both the humoral and T-cell immune response to the 3rd COVID-19 primary vaccine dose in children who are immunocompromised. The results of this study support the utility of the 3rd vaccine dose and the rationale for ongoing emphasis for vaccination against COVID-19 in the immunosuppressed pediatric population., (© 2022. The Author(s), under exclusive licence to the International Pediatric Research Foundation, Inc.)

Technologies that recruit and direct the activity of endogenous RNA-editing enzymes to specific cellular RNAs have therapeutic potential, but translating them from cell culture into animal models has been challenging. Here we describe short, chemically modified oligonucleotides called AIMers that direct efficient and specific A-to-I editing of endogenous transcripts by endogenous adenosine deaminases acting on RNA (ADAR) enzymes, including the ubiquitously and constitutively expressed ADAR1 p110 isoform. We show that fully chemically modified AIMers with chimeric backbones containing stereopure phosphorothioate and nitrogen-containing linkages based on phosphoryl guanidine enhanced potency and editing efficiency 100-fold compared with those with uniformly phosphorothioate-modified backbones in vitro. In vivo, AIMers targeted to hepatocytes with N-acetylgalactosamine achieve up to 50% editing with no bystander editing of the endogenous ACTB transcript in non-human primate liver, with editing persisting for at least one month. These results support further investigation of the therapeutic potential of stereopure AIMers., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Attaining sufficient tissue exposure at the site of action to achieve the desired pharmacodynamic effect on a target is an important determinant for any drug discovery program, and this can be particularly challenging for oligonucleotides in deep tissues of the CNS. Herein, we report the synthesis and impact of stereopure phosphoryl guanidine-containing backbone linkages (PN linkages) to oligonucleotides acting through an RNase H-mediated mechanism, using Malat1 and C9orf72 as benchmarks. We found that the incorporation of various types of PN linkages to a stereopure oligonucleotide backbone can increase potency of silencing in cultured neurons under free-uptake conditions 10-fold compared with similarly modified stereopure phosphorothioate (PS) and phosphodiester (PO)-based molecules. One of these backbone types, called PN-1, also yielded profound silencing benefits throughout the mouse brain and spinal cord at low doses, improving both the potency and durability of response, especially in difficult to reach brain tissues. Given these benefits in preclinical models, the incorporation of PN linkages into stereopure oligonucleotides with chimeric backbone modifications has the potential to render regions of the brain beyond the spinal cord more accessible to oligonucleotides and, consequently, may also expand the scope of neurological indications amenable to oligonucleotide therapeutics., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Classically, endoplasmic reticulum (ER) retention signals in secreted integral membrane proteins impose the requirement to assemble with other cognate subunits to form functional assemblies before they can exit the ER. We report that GluK5 has two ER retention signals in its cytoplasmic C-terminus: an arginine-based signal and a di-leucine motif previously thought to be an endocytic motif. GluK5 assembles with GluK2, but surprisingly GluK2 association does little to block the ER retention signals. We find instead that the ER retention signals are blocked by two proteins involved in intracellular trafficking, SAP97 and CASK. We show that SAP97, in the presence of CASK and the receptor complex, assumes an extended conformation. In the extended conformation, SAP97 makes its SH3 and GuK domains available to bind and sterically mask the ER retention signals in the GluK5 C-terminus. SAP97 and CASK are also necessary for sorting receptor cargoes into the local dendritic secretory pathway in neurons. We show that the ER retention signals of GluK5 play a vital role in sorting the receptor complex in the local dendritic secretory pathway in neurons. These data suggest a new role for ER retention signals in trafficking integral membrane proteins in neurons. SIGNIFICANCE: We present evidence that the ER retention signals in the kainate receptors containing GluK5 impose a requirement for sorting into local dendritic secretory pathways in neurons, as opposed to traversing the somatic Golgi apparatus. There are two ER retention signals in the C-terminus of GluK5. We show that both are blocked by physical association with SAP97 and CASK. The SH3 and GuK domains of SAP97, in the presence of CASK, bind directly to each ER retention signal and form a complex. These results support an entirely new function for ER retention signals in the C-termini of neuronal receptors, such as NMDA and kainate receptors, and define a mechanism for selective entry of receptors into local secretory pathways., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Gated solely by activity-induced changes in intracellular calcium, small-conductance potassium channels (SKs) are critical for a variety of functions in the CNS, from learning and memory to rhythmic activity and sleep. While there is a wealth of information on SK2 gating, kinetics, and Ca(2+) sensitivity, little is known regarding the regulation of SK2 subcellular localization. We report here that synaptic SK2 levels are regulated by the E3 ubiquitin ligase UBE3A, whose deficiency results in Angelman syndrome and overexpression in increased risk of autistic spectrum disorder. UBE3A directly ubiquitinates SK2 in the C-terminal domain, which facilitates endocytosis. In UBE3A-deficient mice, increased postsynaptic SK2 levels result in decreased NMDA receptor activation, thereby impairing hippocampal long-term synaptic plasticity. Impairments in both synaptic plasticity and fear conditioning memory in UBE3A-deficient mice are significantly ameliorated by blocking SK2. These results elucidate a mechanism by which UBE3A directly influences cognitive function., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

SAP97 is directly involved in exporting NMDA receptors with a specific subunit composition from the endoplasmic reticulum (ER). Characterization of the interactions between SAP97 and an NMDA receptor splice variant, GluN1-3, and of the effects on forward trafficking revealed that an ER-level interaction blocked the RXR ER-retention motif in the GluN1-3 cytoplasmic C-terminus in the context of both reporter molecules and full-length receptors. Binding of SAP97 to the PDZ-binding domain of GluN1-3 was required, but the blockade of ER-retention was mediated by the SH3-GuK domains coupled with the action of the N-terminus of SAP97. While other domains of SAP97 were involved in forward trafficking of GluN1-3 out of the ER, the SH3 domain was necessary and sufficient to block the ER retention. This is the first direct evidence for the masking of ER-retention signals by PDZ domain-containing proteins, and provides detailed underlying mechanistic requirements. Such a mechanism could be central to modulating the ER exit of receptors into local, non-conventional or conventional, secretory pathways in neurons., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Förster (or fluorescence) resonance energy transfer (FRET) is a process involving the radiation-less transfer of energy from a "donor" fluorophore to an "acceptor" fluorophore. FRET technology enables the quantitative analysis of molecular dynamics in biophysics and in molecular biology, such as the monitoring of protein-protein interactions, protein-DNA interactions, and protein conformational changes. FRET-based biosensors have been utilized to monitor cellular dynamics not only in heterogeneous cellular populations, but also at the single-cell level in real time. Lately, applications of FRET-based biosensors range from basic biological to biomedical disciplines. Despite the diverse applications of FRET, FRET-based sensors still face many challenges. There is an increasing need for higher fluorescence resolution and improved specificity of FRET biosensors. Additionally, as more FRET-based technologies extend to medical diagnostics, the affordability of FRET reagents becomes a significant concern. Here, we will review current advances and limitations of FRET-based biosensor technology and discuss future FRET applications.

NMDA receptor NR2A/B subunits have PDZ-binding domains on their extreme C-termini that are known to interact with the PSD-95 family and other PDZ proteins. We explore the interactions between PSD-95 family proteins and the NR2A/B cytoplasmic tails, and the consequences of these interactions, from the endoplasmic reticulum (ER) through delivery to the synapse in primary rat hippocampal and cortical cultured neurons. We find that the NR2A/B cytoplasmic tails cluster very early in the secretory pathway and interact serially with SAP102 beginning at the intermediate compartment, and then PSD-95. We further establish that colocalization of the distal C-terminus of NR2B and PSD-95 begins at the trans-Golgi Network (TGN). Formation of NR2B/PSD-95/SAP102 complexes is dependent on the PDZ binding domain of NR2B subunits, but association with SAP102 and PSD-95 plays no distinguishable role in cluster pre-formation or initial targeting to the vicinity of the synapse. Instead the PDZ binding domain plays a role in restricting cell-surface clusters to postsynaptic targets.

Kainate receptors are a class of ionotropic glutamate receptors that are widely expressed in the mammalian brain, yet little is known about their physiological role or the mechanisms by which they are regulated. Kainate receptors are composed of multiple subunits (GluR5-7; KA1-2), which can combine to form homomeric or heteromeric channels. While the kainate receptor subunit KA2 can combine with GluR5-7 to form heteromeric channels, it does not form functional homomeric channels when expressed alone. In an attempt to identify the molecular mechanisms for this, we have characterized the trafficking and surface expression of KA2. We find that KA2 alone does not traffic to the plasma membrane and is retained in the endoplasmic reticulum (ER). In contrast, co-expression with GluR6 disrupts ER-retention of KA2 and allows plasma membrane expression. Using a chimeric reporter protein we have identified an ER-retention motif within the KA2 cytosolic domain. Recent studies have identified a consensus ER-retention motif (RRR) that is contained within both the NMDA receptor NR1 subunit and K(+) channels. While KA2 contains a similar stretch of amino acids within its C-terminus (RRRRR), unlike the NR1 motif, disruption of this motif with alternating glutamic acid residues does not disrupt ER-retention of KA2, suggesting a unique mechanism regulating KA2 surface expression.

The NMDA receptor (NMDAR) plays a central role in the function of excitatory synapses. Recent studies have provided interesting insights into several aspects of the trafficking of this receptor in neurons. The NMDAR is not a static resident of the synapse. Rather, the number and composition of synaptic NMDARs can be modulated by several factors. The interaction of PDZ proteins, generally thought to occur at the synapse, appears to occur early in the secretory pathway; this interaction may play a role in the assembly of the receptor complex and its exit from the endoplasmic reticulum. This review addresses recent advances in our understanding of NMDAR trafficking and its synaptic delivery and maintenance.

Although synaptic AMPA receptors have been shown to rapidly internalize, synaptic NMDA receptors are reported to be static. It is not certain whether NMDA receptor stability at synaptic sites is an inherent property of the receptor, or is due to stabilization by scaffolding proteins. In this study, we demonstrate that NMDA receptors are internalized in both heterologous cells and neurons, and we define an internalization motif, YEKL, on the distal C-terminus of NR2B. In addition, we show that the synaptic protein PSD-95 inhibits NR2B-mediated internalization, and that deletion of the PDZ-binding domain of NR2B increases internalization in neurons. This suggests an involvement for PSD-95 in NMDA receptor regulation and an explanation for NMDA receptor stability at synaptic sites.

We investigated changes in AMPA receptor properties in rat hippocampus 5 h after systemic kainate administration. Quantitative [3H]AMPA autoradiography and Western blot analysis of receptor subunits GluR1-3 in different subcellular fractions were used to evaluate possible alterations in binding characteristics and immunological properties of the receptors in synaptic and nonsynaptic fractions. Both ligand-binding and Western blots revealed significant changes in binding and immunological properties of nonsynaptic receptors but relatively smaller changes in synaptic receptors 5 h after kainate administration. GluR2/3 showed a greater relative change in the synaptic receptor population compared to GluR1, suggesting either a shift in subunit composition of AMPA receptors or the formation of a synaptic subpopulation of AMPA receptors with truncated C-terminal domain of GluR1 subunits. The effects of kainic acid were blocked by cycloheximide treatment indicating that the changes were due at least in part to increased synthesis of AMPA receptor subunits. The results indicate that excessive synaptic activity produces rapid changes in both synaptic and nonsynaptic AMPA receptor properties., (Copyright 1998 Academic Press.)

Quantitative alpha-[3H]amino-3-hydroxy-5-methylisoxazole-4-propionic acid ([3H]AMPA) binding autoradiography was performed on frozen-thawed sections from rat brain after preincubation at 0 or 35 degrees C for 1 h. Preincubation at 35 degrees C instead of 0 degrees C resulted in a selective decrease of [3H]AMPA binding assayed at a low concentration of [3H]AMPA (50 nM) and an enhancement of binding at a high concentration (500 nM). The decrease in [3H]AMPA binding after preincubation at 35 degrees C was accompanied with the loss of the lighter organelles of P3 (microsomal) fractions. These organelles were found to contain a small subpopulation of AMPA/GluR receptors exhibiting a high affinity for [3H]AMPA (K(D) approximately 14 nM), whereas heavier organelles exhibited lower affinity for AMPA (K(D) approximately 190 nM). This small subpopulation of AMPA/GluR receptors contained almost exclusively a structurally distinct species of GluR2/3 subunits with an apparent molecular mass of 103.5 kDa (assessed with anti-GluR2/3, C-terminal antibodies). Experiments using two deglycosylating enzymes, N-glycopeptidase F and endoglycosidase H, clearly indicated that the 103.5-kDa species represented a partially unglycosylated form of GluR2/3 subunits containing the high-mannose type of oligosaccharide moiety, whereas receptors present in synaptosomal fractions were composed of subunits with complex oligosaccharides. A similar result was obtained by using an antibody recognizing the N-terminal domain of GluR2(4). The same enzymatic treatment indicated that GluR1 subunits also exhibited a partially glycosylated form. These data indicate that high-affinity [3H]AMPA binding sites represent nonsynaptic, intracellular membrane-bound AMPA receptors that differ from synaptic receptors by at least the glycosylation state of GluR2 (and GluR1) subunits. In addition, our results provide a relatively simple way of assessing changes in two spatially and structurally distinct [3H]AMPA binding/GluR sites.

Forebrains from rats of postnatal days (PND) 2, 7, 14, 21, and 30-40 were subjected to subcellular fractionation and samples from crude mitochondrial (P2, which contain synaptic plasma membranes) and microsomal (P3) fractions were used for SDS-PAGE and Western blotting with antibodies against GluR1, and GluR2/3 subunits of AMPA/GluR receptors. GluR immunoreactivity in P2 fractions increased gradually from PND 2 to PND 30. In contrast, GluR immunoreactivity in P3 fractions increased sharply at early postnatal ages, and was higher than in adults as early as at PND 7. Data were compared to postnatal changes in 3H-AMPA binding reported in various studies. Significant correlations were observed between changes in GluR immunoreactivity in P3 fractions and changes in high-affinity binding on one hand and between changes in GluR immunoreactivity in P2 fractions, and changes in low affinity binding. These data further establish that glutamate receptors present in different subcellular compartments represent different maturational states of the receptors, and suggest that changes in GluR populations could participate in mechanisms of synaptic plasticity., (Copyright 1998 Elsevier Science B.V.)

The developmental changes in alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor properties in rat hippocampus were evaluated with quantitative autoradiography of ligand binding and in situ hybridization performed in adjacent sections with antisense oligonucleotides for AMPA receptor subunits (GluR1-3, flip and flop splice variants). Specific 3H-AMPA binding in different hippocampal subfields increased between postnatal day 7 and 15 and was higher in CA3 during the postnatal period when compared to adult levels. This effect was mostly due to high levels of high affinity binding sites in cell body layers during the developmental period. By contrast, autoradiograms of 3H-AMPA binding predominantly to the low affinity binding sites indicated an absence of these sites in cell body layers and the overall levels of binding exhibited little overshoot compared to adult levels during the developmental period. The changes in binding of the antagonist of the AMPA receptor, 6-nitro-7-cyanoquinoxaline-2,3-dione were markedly different from those for the high affinity AMPA binding sites but quite similar to those for the low affinity sites. The binding was extremely low at postnatal day 7 and increased rapidly between postnatal day 7 and 15 and slowly between postnatal day 15 and adult. Low levels of binding were observed in the cell body layer at every postnatal age. The changes in expression of messenger RNAs for the different subunits of the AMPA receptors were well correlated with the modifications in high affinity AMPA binding sites measured in the cell body layers also exhibiting an increased expression of the receptors at the transcriptional level during the developmental period as compared to adult levels. The relative expression of the GluR2 subunits decreased during the postnatal period and the time course for this reduction paralleled that for the increased vulnerability of hippocampal pyramidal neurons to a variety of insults. The results indicate that both the messenger RNAs for the subunits and the AMPA receptors exhibit increased levels of expression during the postnatal period compared to adult levels. They also suggest that nascent receptors might bind AMPA with high affinity before their insertion in membranes into functional receptors that have low affinity for agonists and high affinity for antagonists. The changes in subunit composition of the receptors during the postnatal period may have important implications for mechanisms of plasticity as well as of neuropathology.

The distribution of glutamate AMPA receptors in the synaptosomal and microsomal fractions of neonatal and adult rat telencephalon was studied by determining the saturation kinetics at equilibrium of 3H-AMPA and 3H-CNQX binding. At both ages, synaptosomal preparations exhibited two populations of 3H-AMPA binding sites with a small number of high affinity sites and a large number of low affinity sites. 3H-AMPA binding to microsomal preparations from both neonatal and adult rat telencephalon exhibited a much higher proportion of high affinity relative to low affinity sites. 3H-CNQX binding to the same fractions did not parallel 3H-AMPA binding, but was correlated with the low affinity 3H-AMPA binding and with a marker of plasma membranes. The results suggest that nonsynaptic glutamate/AMPA receptors have a high affinity for agonist and become low affinity when inserted into postsynaptic membranes and that 3H-CNQX binds synaptic but not nonsynaptic glutamate/AMPA receptors with high affinity.

The expression of long-term potentiation and learning of a classical conditioning task increase [3H]-AMPA binding in hippocampus. Phospholipase A2 (PLA2) has been proposed to underly these changes, as PLA2 treatment of membrane preparations increases the affinity of AMPA receptors for agonists. We demonstrate here that preincubation of thin (10 microns) frozen rat brain sections with exogenous PLA2 and calcium at physiological temperature changes the binding properties of AMPA receptors. Quantitative autoradiography reveals that PLA2-treatment produces a differential increase in [3H]-AMPA binding across brain regions. The same treatment also decreases the binding of an antagonist ([3H]-CNQX) throughout the brain. We propose that PLA2 treatment results in a modification of the AMPA receptors which is regionally specific, probably due to different AMPA receptor subunit compositions.