Daniel P. Morris

Phospho-carboxyl-terminal domain binding and the role of a prolyl isomerase in pre-mRNA 3'-End formation.

A phospho-carboxyl-terminal domain (CTD) affinity column created with yeast CTD kinase I and the CTD of RNA polymerase II was used to identify Ess1/Pin1 as a phospho-CTD-binding protein. Ess1/Pin1 is a peptidyl prolyl isomerase involved in both mitotic regulation and pre-mRNA 3′-end formation. Like native Ess1, a GSTEss1 fusion protein associates specifically with the phosphorylated but not with the unphosphorylated CTD. Further, hyperphosphorylated RNA polymerase II appears to be the dominant Ess1 binding protein in total yeast extracts. We demonstrate that phospho-CTD binding is mediated by the small WW domain of Ess1 rather than the isomerase domain. These findings suggest a mechanism in which the WW domain binds the phosphorylated CTD of elongating RNA polymerase II and the isomerase domain reconfigures the CTD though isomerization of proline residues perhaps by a processive mechanism. This process may be linked to a variety of pre-mRNA maturation events that use the phosphorylated CTD, including the coupled processes of pre-mRNA 3′-end formation and transcription termination.

Epigenetic regulation of human α1d-adrenergic receptor gene expression: a role for DNA methylation in Sp1-dependent regulation

A growing body of evidence implicates α1‐adrenergic receptors (α1ARs) as potent regulators of growth pathways. The three α1AR subtypes (α1aAR, α1bAR, α1dAR) display highly restricted tissue expression that undergoes subtype switching with many pathological stimuli, the mechanistic basis of which remains unknown. To gain insight into transcriptional pathways governing cell‐specific regulation of the human α1dAR subtype, we cloned and characterized the α1dAR promoter region in two human cellular models that display disparate levels of endogenous α1dAR expression (SK‐N‐MC and DU145). Results reveal that α1dAR basal expression is regulated by Sp1‐dependent binding of two promoter‐proximal GC boxes, the mutation of which attenuates α1dAR promoter activity 10‐fold. Mechanistically, chromatin immunoprecipitation data demonstrate that Sp1 binding correlates with expression of the endogenous gene in vivo, correlating highly with α1dAR promoter methylation‐dependent silencing of both episomally expressed reporter constructs and the endogenous gene. Further, analysis of methylation status of proximal GC boxes using sodium bisulfite sequencing reveals differential methylation of proximal GC boxes in the two cell lines examined. Together, the data support a mechanism of methylation‐depen‐dent disruption of Sp1 binding in a cell‐specific manner resulting in repression of basal α1dAR expression.–Michelotti, G. A., Brinkley, D. M., Morris, D. P., Smith, M. P., Louie, R. J., Schwinn, D. A. Epigenetic regulation of human α1d‐adrenergic receptor gene expression: a role for DNA methylation in Sp1‐dependent regulation. FASEB J. 21, 1979–1993 (2007)

Novel human α1a-adrenoceptor single nucleotide polymorphisms alter receptor pharmacology and biological function

We identified nine naturally-occurring human single nucleotide polymorphisms (SNPs) in the α1a-adrenoceptor (α1aAR) coding region, seven of which result in amino acid change. Utilizing rat-1 fibroblasts stably expressing wild type α1aAR or each SNP at both high and low levels, we investigated the effect of these SNPs on receptor function. Compared with wild type, two SNPs (R166K, V311I) cause a decrease in binding affinity for agonists norepinephrine, epinephrine, and phenylephrine, and also shift the dose–response curve for norepinephrine stimulation of inositol phosphate (IP) production to the right (reduced potency) without altering maximal IP activity. In addition, SNP V311I and I200S display altered antagonist binding. Interestingly, a receptor with SNP G247R (located in the third intracellular loop) displays increased maximal receptor IP activity and stimulates cell growth. The increased receptor signaling for α1aAR G247R is not mediated by altered ligand binding or a deficiency in agonist-mediated desensitization, but appears to be related to enhanced receptor–G protein coupling. In conclusion, four naturally-occurring human α1aAR SNPs induce altered receptor pharmacology and/or biological activity. This finding has potentially important implications in many areas of medicine and can be used to guide α1aAR SNP choice for future clinical studies.

The α1a-Adrenergic Receptor Occupies Membrane Rafts with Its G Protein Effectors but Internalizes via Clathrin-coated Pits

The α1a-adrenergic receptor (α1aAR) occupies intracellular and plasma membranes in both native and heterologous expression systems. Based on multiple independent lines of evidence, we demonstrate the α1aAR at the cell surface occupies membrane rafts but exits from rafts following stimulation. In non-detergent raft preparations, basal α1aAR is present in low density membrane rafts and colocalizes with its G protein effectors on density gradients. Raft disruption by cholesterol depletion with methyl-β-cyclodextrin eliminates these light rafts. To confirm the presence of the α1aAR in plasma membrane rafts, fluorescence resonance energy transfer measurements were used to demonstrate colocalization of surface receptor and the raft marker, cholera toxin B. This colocalization was largely lost following α1aAR stimulation with phenylephrine. Similarly, receptor stimulation causes exit of the α1aAR from light rafts within 3-10 min in contrast to the G proteins, which largely remain in light rafts. Importantly, this delayed exit of the α1aAR suggests acute receptor signaling and desensitization occur entirely within rafts. Interestingly, both confocal analysis and measurement of surface α1aAR levels indicate modest receptor internalization during the 10 min following stimulation, suggesting most of the receptor has entered non-raft plasma membrane. Nevertheless, activation does increase the rate of receptor internalization as does disruption of rafts with methyl-β-cyclodextrin, suggesting raft exit enables internalization. Confocal analysis of surface-labeled hemagglutinin-α1aAR reveals that basal and stimulated receptor occupies clathrin pits in fixed cells consistent with previous indirect evidence. The evidence presented here strongly suggests the α1aAR is a lipid raft protein under basal conditions and implies agonist-mediated signaling occurs from rafts.

Kinetic characterization of Salmonella FliK-FlhB interactions demonstrates complexity of the Type III secretion substrate-specificity switch.

The bacterial flagellum is a complex macromolecular machine consisting of more than 20 000 proteins, most of which must be exported from the cell via a dedicated Type III secretion apparatus. At a defined point in flagellar morphogenesis, hook completion is sensed and the apparatus switches substrate specificity type from rod and hook proteins to filament ones. How the switch works is a subject of intense interest. FliK and FlhB play central roles. In the present study, two optical biosensing methods were used to characterize FliK-FlhB interactions using wild-type and two variant FlhBs from mutants with severe flagellar structural defects. Binding was found to be complex with fast and slow association and dissociation components. Surprisingly, wild-type and variant FlhBs had similar kinetic profiles and apparent affinities, which ranged between 1 and 10.5 microM, suggesting that the specificity switch is more complex than presently understood. Other binding experiments provided evidence for a conformational change after binding. Liquid chromatography-mass spectrometry (LC-MS) and NMR experiments were performed to identify a cyclic intermediate product whose existence supports the mechanism of autocatalytic cleavage at FlhB residue N269. The present results show that while autocatalytic cleavage is necessary for proper substrate specificity switching, it does not result in an altered interaction with FliK, strongly suggesting the involvement of other proteins in the mechanism.

Lipid rafts constrain basal α1A-adrenergic receptor signaling by maintaining receptor in an inactive conformation

We have reported that the alpha(1A)-adrenergic receptor (alpha(1A)AR) in rat-1 fibroblasts is a lipid raft protein. Here we examined whether disrupting lipid rafts by methyl-beta-cyclodextrin (MCD) sequestration of cholesterol affects alpha(1A)AR signaling. Unexpectedly, MCD increased alpha(1A)AR-dependent basal inositol phosphate formation and p38 mitogen-activated protein kinase activation in a cholesterol-dependent manner. It also initiated internalization of surface alpha(1A)AR, which was partially blocked by receptor inhibition. Binding assays revealed MCD-mediated increases in receptor agonist affinity as well as reciprocal decreases in inverse agonist affinity, a behavior that is usually interpreted as a shift toward the active receptor conformation. In untreated cells a fraction of the receptor was found to be present in preassociated receptor/G protein complexes, which rapidly dissociate upon receptor stimulation. Consistent with MCD-induced signaling, raft disruption resulted in an increase in receptor/G protein complexes. These results strongly suggest that lipid rafts constrain basal alpha(1A)AR activity; however, preassembled receptor/G protein complexes could still provide a mechanism for accelerating alpha(1A)AR signaling following stimulation.

The active site region of the vitamin K-dependent carboxylase includes both the amino-terminal hydrophobic and carboxy-terminal hydrophilic domains of the protein

In order to localize the active site of the vitamin K‐dependent carboxylase, we developed an affinity probe containing the propeptide and the first two carboxylatable glutamate residues conserved in many native substrates. This probe crosslinked to both the hydrophobic amino‐terminal and hydrophilic carboxy‐terminal domains of the carboxylase, in contrast with previous work which localized both the catalytic and the propeptide binding site within the amino‐terminal hydrophobic domain. Amino acid analysis revealed that the mass of an amino‐terminal fragment is seriously underestimated by SDS‐PAGE. Reanalysis of the published data in light of this information suggests that a portion of the propeptide binding site resides within the carboxy‐terminal hydrophilic domain.

Temporal Dissection of Rate Limiting Transcriptional Events Using Pol II ChIP and RNA Analysis of Adrenergic Stress Gene Activation

In mammals, increasing evidence supports mechanisms of co-transcriptional gene regulation and the generality of genetic control subsequent to RNA polymerase II (Pol II) recruitment. In this report, we use Pol II Chromatin Immunoprecipitation to investigate relationships between the mechanistic events controlling immediate early gene (IEG) activation following stimulation of the α1a-Adrenergic Receptor expressed in rat-1 fibroblasts. We validate our Pol II ChIP assay by comparison to major transcriptional events assessable by microarray and PCR analysis of precursor and mature mRNA. Temporal analysis of Pol II density suggests that reduced proximal pausing often enhances gene expression and was essential for Nr4a3 expression. Nevertheless, for Nr4a3 and several other genes, proximal pausing delayed the time required for initiation of productive elongation, consistent with a role in ensuring transcriptional fidelity. Arrival of Pol II at the 3’ cleavage site usually correlated with increased polyadenylated mRNA; however, for Nfil3 and probably Gprc5a expression was delayed and accompanied by apparent pre-mRNA degradation. Intragenic pausing not associated with polyadenylation was also found to regulate and delay Gprc5a expression. Temporal analysis of Nr4a3, Dusp5 and Nfil3 shows that transcription of native IEG genes can proceed at velocities of 3.5 to 4 kilobases/min immediately after activation. Of note, all of the genes studied here also used increased Pol II recruitment as an important regulator of expression. Nevertheless, the generality of co-transcriptional regulation during IEG activation suggests temporal and integrated analysis will often be necessary to distinguish causative from potential rate limiting mechanisms.