Signal transduction through trimeric G proteins in megakaryoblastic cell lines.

The biogenesis of trimeric G proteins was investigated by measurement of the expression of alpha-subunits in the megakaryoblastic cell lines MEG-01, DAMI, and CHRF-288-11, representing stages of increasing maturation, and compared with platelets. Megakaryoblasts and platelets contained approximately equal amounts of Gi alpha-1/2, Gi alpha-3, Gq alpha, and G12 alpha protein. Maturation was accompanied by (1) downregulation of mRNA for Gs alpha and disappearance of iloprost-induced Ca2+ mobilization, (2) upregulation of the long form of Gs alpha protein (Gs alpha-L) and an increase in iloprost-induced cAMP formation, and (3) upregulation of G16 alpha mRNA and G16 alpha protein and appearance of thromboxane A2-induced signaling (Ca2+ mobilization and stimulation of prostaglandin I2-induced cAMP formation). Gz alpha protein was absent in the megakaryoblasts despite weak expression of Gz alpha mRNA in DAMI and relatively high levels of Gz alpha mRNA and Gz alpha protein in platelets. These findings reveal major changes in G protein-mediated signal transduction during megakaryocytopoiesis and indicate that G16 alpha couples the thromboxane receptor to phospholipase C beta.

The promoter and intron/exon structure of the human and mouse beta 3-adrenergic-receptor genes.

Transcription-start sites for the mouse and human beta 3-adrenergic-receptor mRNA have been localized in a region comprised between 150 and 200 nucleotides 5' from the ATG translation-start codon. Motifs potentially implicated in heterologous regulation of beta 3-adrenergic-receptor expression by glucocorticoids and by beta-adrenergic agonists have been identified upstream from these cap sites. In mouse, a second mRNA initiation region is postulated to exist further upstream. Comparison of the nucleotide sequences of the 3' end of the human and mouse beta 3-adrenergic-receptor genes to those of the corresponding cDNA revealed that in contrast to beta 1 and beta 2 adrenergic receptors, the beta 3-adrenergic-receptor genes comprise several exons. A large exon (1.4 kb) encodes the first 402 and 388 amino-acid residues of the human and mouse beta 3 adrenergic receptor, respectively. In man, a second exon (700 bp) contains the sequence coding for the six carboxy-terminal residues of the receptor and the entire mRNA 3' untranslated region. In mouse, a second exon (68 bp) codes for the 12 carboxy-terminal residues of the receptor and a third exon contains the beta 3-adrenergic-receptor mRNA 3' untranslated region. The use of alternate acceptor splice sites generates two forms of exon 3 (600 bp and 700 bp), yielding two beta 3-adrenergic-receptor transcripts which are differentially expressed in white and brown adipose tissues. Human beta 3-adrenergic-receptor transcripts with different 3' untranslated regions are produced by continuation of transcription beyond termination signals. Together, our results suggest that utilization of alternate promoters and/or 3' untranslated regions may allow tissue-specific regulation of beta 3-adrenergic-receptors expression.

Tissue distribution of beta 3-adrenergic receptor mRNA in man.

Expression of mRNA for beta 1-, beta 2-, and beta 3-adrenergic receptors (beta 1-, beta 2-, and beta 3-AR) was investigated in human tissues. beta 1- and beta 2-AR mRNA distribution correlated with that of the cognate receptors established by pharmacological studies. beta 3-AR transcripts were abundant in infant perirenal brown adipose tissue, characterized by the presence of uncoupling protein (UCP) mRNA. In adult whole adipose tissues, beta 3-AR mRNA levels were high in deep deposits such as perirenal and omental, and lower in subcutaneous. In these deposits, UCP mRNA levels paralleled those of beta 3-AR. However, isolated omental and subcutaneous adipose cells, enriched in white adipocytes, expressed beta 3-AR but no UCP transcripts. beta 3-AR mRNA was highly expressed in gallbladder, and to a much lower extent in colon, independently of UCP mRNA. Quadriceps or abdominal muscles, heart, liver, lung, kidney, thyroid, and lymphocytes did not express intrinsic beta 3-AR mRNA. This study demonstrates that substantial amounts of brown adipocytes exist throughout life in adipose deposits, which are generally classified as white. These deposits are the main sites of beta 3-AR expression, which also occurs in gallbladder and colon. beta 3-AR may thus be involved in the control of lipid metabolism, possibly from fat assimilation in the digestive tract, to triglyceride storage and mobilization in adipose tissues.

Molecular characterization of a human anti-Rh(D) antibody with a DH segment encoded by a germ-line sequence.

The lambda-light-chain and lambda-heavy-chain variable-region genes of an anti-Rh(D) (Rh, Rhesus; D, heavy-chain diversity region) human monoclonal antibody secreted by lymphocytes transformed by the Epstein-Barr virus have been cloned and sequenced. Sequence comparison of the anti-Rh(D)mAb lambda-chain variable region with those of the other available human lambda chains revealed that it belonged to the human V lambda I (V lambda, variable region of lambda chain) subgroup. The greatest sequence similarity (80%) was observed with that of another anti-Rh antibody lambda-chain directed against the Rh(c) antigen. For the VH (VH, variable region of heavy chain) sequence, the highest similarity (86%) was observed with the germline VHG3 gene which belongs to the VHI subgroup. The expressed DH sequence of the anti-Rh(D) antibody is also of germline origin and complementarity-determining region 3 is thus produced by VH-DH and DH-JH (J, joining region) joining without recombination of multiple DH gene segments.