Numerous mutations in the humancomprising the major structural components of the nuclear lamina, the fibrous meshwork underlying the inner nuclear membrane (1). They are major determinants of nuclear size and shape and are involved in essential functions such as DNA replication and transcription (1). Lamins A (LA) and C (LC) are alternatively spliced products of the LMNA gene, whereas lamins LB1 and LB2 are encoded by LMNB1 and LMNB2. Structurally, the lamins have a conserved central ␣-helical rod domain flanked by globular head and tail domains. The central rod is responsible for the formation of in-parallel and in-register coiled-coil dimers, the building blocks of lamin polymers. In vitro, lamin dimers form head-to-tail chains, which further interact laterally in an anti-parallel orientation to form highly ordered paracrystals (PCs) (2). However, little is known about the assembly of lamins into higher order structures in vivo.There are Ͼ250 mutations in human LMNA causing a wide range of diseases [for detail, see http//www.umd.be/LMNA/ and (3)]. Among these is the rare premature aging disease, Hutchinson-Gilford progeria syndrome (HGPS). HGPS children typically appear normal at birth, but show growth retardation before the age of 2 years. Further manifestations include loss of hair, lipodystrophy, sclerodermatous skin, osteolysis, and progressive atherosclerosis leading to death at an average age of 13 years due to myocardial infarcts and strokes (4). Most HGPS patients carry the 1824CϾT mutation (G608G), which activates a cryptic splice site resulting in the expression of LA with 50 amino acids deleted near its C terminus (LA⌬50/progerin) (5). As a result, LA⌬50/ progerin remains permanently farnesylated (6, 7), and its accumulation in patients' cells is correlated with the loss of heterochromatin and changes in histone methylation (1).In addition to 1824CϾT, there are 21 other LMNA mutations causing progeria (http://www.umd.be/LMNA/). Some of these are located in the central rod domain, where they could directly impact the assembly of lamins into dimers and higher order structures. Here, we have studied the alterations in nuclear architecture and chromatin organization in one of these mutations, 433GϾA (E145K), located in segment 1B of the central rod domain of LA/C (5). A patient bearing this mutation showed earlier onset cardiovascular defects, only partial loss of hair and ample s.c. fat (5). We show that fibroblasts derived from an E145K patient have severely misshapen nuclei and multiple defects in chromatin organization as reflected by centromere clustering and an abnormal distribution of telomeres throughout the cell cycle. These abnormalities are established during cell division as nuclei assemble in daughter cells. The results demonstrate that the nuclear changes in E145K progeria cells are significantly different from those seen in cells expressing LA⌬50/ progerin, and they also emphasize the essential role of lamins in establishing and maintaining nuclear architecture.

Within the family of serotonin receptors, the 5-hydroxytryptamine-3 (5-HT 3 ) receptor is the only ligand-gated ion channel. It is composed of five subunits, of which the 5-HT 3A and 5-HT 3B subunits are best characterized. Several studies, however, have reported on the functional diversity of native 5-HT 3 receptors, which cannot solely be explained on the basis of the 5-HT 3A and 5-HT 3B subunits. After our discovery of further putative 5-HT 3 serotonin receptor-encoding genes, HTR3C, HTR3D, and HTR3E, we investigated whether these novel candidates and the isoform 5-HT 3Ea are able to form functional 5-HT 3 receptor complexes. Using immunofluorescence and immunoprecipitation studies of heterologously expressed proteins, we found that each of the respective candidates coassembles with 5-HT 3A . To investigate whether the novel subunits modulate 5-HT 3 receptor function, we performed radioligandbinding assays and calcium-influx studies in human embryonic kidney 293 cells. Our experiments revealed that the 5-HT 3C , 5-HT 3D , 5-HT 3E , and 5-HT 3Ea subunits alone cannot form functional receptors. Coexpression with 5-HT 3A , however, results in the formation of functional heteromeric complexes with different serotonin efficacies. Potencies of two agonists and antagonists were nearly identical with respect to homomeric 5-HT 3A and heteromeric complexes. However, 5-HT showed increased efficacy with respect to 5-HT 3A/D and 5-HT 3A/E receptors, which is consistent with the increased surface expression compared with 5-HT 3A receptors. In contrast, 5-HT 3A/C and 5-HT 3A/Ea receptors exhibited decreased 5-HT efficacy. These data show for the first time that the novel 5-HT 3 subunits are able to form heteromeric 5-HT 3 receptors, which exhibit quantitatively different functional properties compared with homomeric 5-HT 3A receptors.The 5-HT 3 receptor is the only ligand-gated ion channel (LGIC) within the family of serotonin (5-hydroxytryptamine, 5-HT) receptors (Hoyer et al., 2002). Based on structural and functional homologies, the nicotinic acetylcholine receptor and the 5-HT 3 receptor are most closely related; both are cation channels. The 5-HT 3 receptor is formed by a pentameric complex and is permeable to Na ϩ , K ϩ , and Ca 2ϩ . Binding of serotonin to the 5-HT 3 receptor leads to a fast excitatory response of the neuron. After cloning of the human HTR3A gene (Belelli et al., 1995;Miyake et al., 1995), findings concerning variable receptor compositions and properties led to the hypothesis that further 5-HT 3 receptor subunits and isoforms should exist (Hussy et al., 1994;Jackson and Yakel, 1995;Fletcher and Barnes, 1998). This hypothesis was confirmed by the cloning of the human HTR3B gene (Davies et al., 1999) and of two different human splice variants of the HTR3A gene (Brü ss et al., 2000). To date, HTR3A and HTR3B (Belelli et al., 1995;Miyake et al., 1995;Davies et al., 1999) are well characterized. 5-HT 3A subunits are able to form functional homooligomeric receptors after expression in Xenopus laevi...

Diarrhea predominant irritable bowel syndrome (IBS-D) is a complex disorder related to dysfunctions in the serotonergic system. As cis-regulatory variants can play a role in the etiology of complex conditions, we investigated the untranslated regions (UTRs) of the serotonin receptor type 3 subunit genes HTR3A and HTR3E. Mutation analysis was carried out in a pilot sample of 200 IBS patients and 100 healthy controls from the UK. The novel HTR3E 3'-UTR variant c.*76G>A (rs62625044) was associated with female IBS-D (P = 0.033, OR = 8.53). This association was confirmed in a replication study, including 119 IBS-D patients and 195 controls from Germany (P = 0.0046, OR = 4.92). Pooled analysis resulted in a highly significant association of c.*76G>A with female IBS-D (P = 0.0002, OR = 5.39). In a reporter assay, c.*76G>A affected binding of miR-510 to the HTR3E 3'-UTR and caused elevated luciferase expression. HTR3E and miR-510 co-localize in enterocytes of the gut epithelium as shown by in situ hybridization and RT-PCR. This is the first example indicating micro RNA-related expression regulation of a serotonin receptor gene with a cis-regulatory variant affecting this regulation and appearing to be associated with female IBS-D.

Dopamine D2 receptor-promoted activation of Gα(o) over Gα(i) may increase synaptic plasticity and thereby might improve negative symptoms of schizophrenia. Heterocyclic dopamine surrogates comprising a pyrazolo[1,5-a]pyridine moiety were synthesized and investigated for their binding properties when low- to subnanomolar K(i) values were determined for D(2L), D(2S), and D3 receptors. Measurement of [(35)S]GTPγS incorporation at D(2S) coexpressed with G-protein subunits indicated significant bias for promotion of Gα(o1) over Gα(i2) coupling for several test compounds. Functionally selective D(2S) activation was most striking for the carbaldoxime 8b (Gα(o1), pEC50 = 8.87, E(max) = 65%; Gα(i2), pEC50 = 6.63, E(max) = 27%). In contrast, the investigated 1,4-disubstituted aromatic piperazines (1,4-DAPs) behaved as antagonists for β-arrestin-2 recruitment, implying significant ligand bias for G-protein activation over β-arrestin-2 recruitment at D(2S) receptors. Ligand efficacy and selectivity between D(2S) and D3 activation were strongly influenced by regiochemistry and the nature of functional groups attached to the pyrazolo[1,5-a]pyridine moiety.

Crystal structures of G protein-coupled receptor (GPCR) ligand complexes allow a rational design of novel molecular probes and drugs. Here we report the structure-guided design, chemical synthesis and biological investigations of bivalent ligands for dopamine D2 receptor/neurotensin NTS1 receptor (D2R/NTS1R) heterodimers. The compounds of types 1–3 consist of three different D2R pharmacophores bound to an affinity-generating lipophilic appendage, a polyethylene glycol-based linker and the NTS1R agonist NT(8-13). The bivalent ligands show binding affinity in the picomolar range for cells coexpressing both GPCRs and unprecedented selectivity (up to three orders of magnitude), compared with cells that only express D2Rs. A functional switch is observed for the bivalent ligands 3b,c inhibiting cAMP formation in cells singly expressing D2Rs but stimulating cAMP accumulation in D2R/NTS1R-coexpressing cells. Moreover, the newly synthesized bivalent ligands show a strong, predominantly NTS1R-mediated β-arrestin-2 recruitment at the D2R/NTS1R-coexpressing cells.