Signal transduction via G-protein-linked receptors: physiological regulation from the plasma membrane to the genome.

Many neurotransmitters, hormones, and drugs express their actions through binding to cell-surface receptors that are coupled to membrane-localized effectors via GTP-binding regulatory proteins (G-proteins). Muscarinic acetylcholine, alpha- and beta-adrenergic receptors are members of this populous class of G-protein-linked receptors. Adenylyl cyclase, phospholipase C, and ion channel activities are examples of effectors regulated via these receptors. Signal transduction via G-protein-linked receptors can be regulated at the level of the receptor, G-protein(s), and effector(s). Activation of G-protein-mediated pathway propagates the signal and leads to desensitization (short-term adaptation) and then down-regulation (long-term adaptation). How transmembrane signaling is linked to expression at the level of the gene (transcriptional control), at the level of mRNA (post-transcriptional control) and at the level of the protein (post-translational modification) remains a central question of neurobiology. Investigations at each of these potential loci for regulation have begun to reveal the molecular basis for down-regulation by agonist, up-regulation by permissive hormones (like adrenal steroids), and cross-regulation among G-protein-mediated pathways. The general topic will be discussed drawing upon recent studies of the regulation of the adrenergic receptor family (alpha- and beta-). These recent advances provide a focus for a broader understanding of the integration of information between the genome and transmembrane signaling.

Beta 1- and beta 2-adrenergic receptor expression in differentiating 3T3-L1 cells. Independent regulation at the level of mRNA.

Regulation of two highly homologous GTP-binding regulatory protein- (G-protein) linked receptors, beta 1- and beta 2-adrenergic receptors, was probed at the level of mRNA in differentiating 3T3-L1 cells. Expression of the two receptor subtypes at the protein level was defined by competition of radioligand binding with CGP-20712A, a highly selective beta 1-adrenergic antagonist. 3T3-L1 fibroblasts express equivalent levels of beta 1- and beta 2-adrenergic receptors. Following treatment with dexamethasone and isobutylmethyl xanthine (IBMX), 3T3-L1 cells differentiate to adipocytes and express 4-fold more receptor, predominantly beta 2-subtype (beta 1-/beta 2- ratio, 5:95). Regulation of beta 1- and beta 2-receptor mRNA levels by differentiation, as well as by steroid alone and IBMX alone was probed by DNA excess solution hybridization. A beta 1-receptor antisense probe was constructed from double-stranded DNA assembled from synthetic oligonucleotides. In untreated 3T3-L1 fibroblasts the steady-state levels of beta 1- and beta 2-adrenergic receptor mRNA were equivalent (approximately 1.2 amol mRNA/micrograms total cellular RNA). beta 2-Adrenergic receptor mRNA levels increased 3-fold as 3T3-L1 fibroblasts were differentiated to adiopcytes (day 7). mRNA levels for beta 1-adrenergic receptor, in contrast, increased at day 2, but thereafter declined, falling to less than 0.05 amol mRNA/micrograms total cellular RNA by day 7 in adipocytes. A 7-day challenge with dexamethasone reduced by 50% beta 1-adrenergic receptor mRNA levels. Treatment with IBMX alone reduced mRNA levels for both receptor subtypes. Neither steroid nor IBMX alone promoted differentiation. The present work, for the first time, demonstrates (i) the mRNA levels on a molar basis for two highly homologous G-protein-linked receptors expressed in a single cell, (ii) independent regulation of their mRNA levels that correlates well with receptor expression, and (iii), that it is differentiation in 3T3-L1 cells per se and not treatment with glucocorticoid or IBMX alone that promotes the up-regulation of the beta 2-receptor transcripts and down-regulation of beta 1-receptor transcripts.

Team physician #7. A comparative study of functional bracing in the anterior cruciate deficient knee.

We evaluated the ability of three functional knee braces, (CTI, OTI, and TS7) to control anterolateral rotary instability of the knee. Fourteen subjects, none of elite athletic status, with arthroscopically proven anterior cruciate deficient knees were selected. The subjects evaluated each brace after one-month periods, and then underwent testing with physical examinations, KT-1000 arthrometry, and timed running events. All braces reduced subjective symptoms of knee instability. Different subjects preferred different braces. KT-1000 testing showed a reduction in anterior tibial displacement for all braces; however, this reduction did not increase as forces increased. A timed figure-of-eight running event did not show any functional advantage of bracing. Five subluxation events occurred in four subjects while braced. Functional braces appear to have a role in the anterior cruciate deficient knee, but only in conjunction with activity modification.