A threonine to isoleucine polymorphism at amino acid 164 in the fourth transmembrane spanning domain of the 182-adrenergic receptor (,B2AR) is known to occur in the human population. The The human heart expresses both the ,i3AR and f32AR subtypes (6). Each receptor mediates positive inotropic and chronotropic responses to endogenous catecholamines and exogenously administered agonists (7). Most forms of congestive heart failure, including the "idiopathic" forms, are characterized by a diminished responsiveness to 13-agonists (8). As a probable consequence of elevated catecholamines (and perhaps of other factors as well), J31AR expression has been noted to be markedly reduced in patients with idiopathic dilated cardiomyopathy, a finding that has not been observed for the 32AR (8). The 132AR, then, may take on an even greater role in providing for cardiac responses to increased sympathetic drive or exogenous agonists under these circumstances. We have considered that if the Ile-164 variant is in fact dysfunctional in the heart, individuals harboring this receptor with heart failure may exhibit marked decompensation.The current study was undertaken to explore the relevance of the Ile-164 receptor in cardiac myocyte signaling at both the cellular and the whole organ levels. The strategy was to express in transgenic mice the human wild-type (32AR (wt 832AR) or the Ile-164 variant in a cardiac specific manner using the murine a myosin heavy chain (aMHC) promoter. In vitro and in vivo assessments of receptor function were then undertaken that revealed a substantial impairment imposed by this polymorphism in cellular as well as intact heart function. METHODSTransgenic Mice. Cardiac-specific expression of the 32AR in transgenic mice was achieved using the murine aMHC promoter (9). Briefly, the coding block of human wt 132AR cDNA was blunt-end ligated into an aMHC promoter construct (9) at the Sall site. To generate the Ile-164 polymorphism, oligonucleotide-directed site-specific mutagenesis was carried out on the wild-type template, substituting thymidine for cytidine at nucleic acid 491 as described (4). This mutated cDNA was also cloned into the aMHC SailI site, and the integrity of the two constructs was verified by dideoxy sequencing. Each was then digested by NotI and the liberated fragment was then isolated, purified, and used for injection into male pronuclei of embryos

Tissue type transglutaminase (TGII, also known as G h ) has been considered a multifunctional protein, with both transglutaminase and GTPase activity. The role of the latter function, which is proposed as a coupling mechanism between ␣ 1 -adrenergic receptors and phospholipase C (PLC), is not well defined. TGII was overexpressed in transgenic mice in a cardiac specific manner to delineated relevant signaling pathways and their consequences in the heart. Cardiac transglutaminase activity in the highest expressing line was ϳ37-fold greater than in nontransgenic lines. However, in vivo signaling to PLC, as assessed by inositol phosphate turnover in [ 3 H]myoinositol organ bath atrial preparations, was not increased in the TGII mice at base line or in response to ␣ 1 -adrenergic receptor stimulation; nor was protein kinase C␣ (PKC␣) or PKC⑀ activity enhanced in the TGII transgenic mice. This is in contrast to mice moderately (ϳ5-fold) overexpressing G ␣q , where inositol phosphate turnover and PKC activity were found to be clearly enhanced. TGII overexpression resulted in a remodeling of the heart with mild hypertrophy, elevated expression of ␤-myosin heavy chain and ␣-skeletal actin genes, and diffuse interstitial fibrosis. Resting ventricular function was depressed, but responsiveness to ␤-agonist was not impaired. This set of pathophysiologic findings is distinct from that evoked by overexpression of G ␣q . We conclude that TGII acts in the heart primarily as a transglutaminase, and modulation of this function results in unique pathologic sequelae. Evidence for TGII acting as a G-protein-like transducer of receptor signaling to PLC in the heart is not supported by these studies.Tissue type transglutaminase (TGII, 1 also known as G h ) is a multifunctional GTP-binding protein that has been proposed to mediate both transglutamination and receptor-stimulated PLC activation (1, 2). As a transglutaminase, TGII catalyzes Ca 2ϩ -dependent post-translational modification of proteins through formation of isopeptide bonds between glutamine and lysine residues (3). However, unlike other transglutaminases, TGII binds guanine nucleotides in a 1:1 ratio and hydrolyzes GTP (4,5). This GTPase activity is independent of transglutaminase activity (6) and has been shown to mediate ␣ 1 -adrenergic receptor (␣ 1 AR) stimulation of phospholipase C ␦1, increasing inositol phosphate turnover in TGII-transfected cells (7-13).Numerous in vitro studies implicate TGII in a wide variety of biological processes, including regulation of cell growth and differentiation (14 -16), apoptosis (17), and tissue repair (18,19), as well as signal transduction via ␣ 1 AR. The role of TGII, however, in regulation of these events in relevant target tissues remains unclear. Activation of ␣ 1 AR expressed on myocytes by catecholamines stimulates PLC activity, increasing inositol phosphate turnover in the heart (20). Traditionally, this signal has been considered to be transduced by the ␣ subunit of the heterotrimeric G protein G q (21). Indeed, a number of ...

Two corticotropin-releasing factor 2 receptor (CRF2R)-selective peptides have been recently described, urocortin II (also known as stresscopin-related peptide) and urocortin III (stresscopin). We have used urocortin II to evaluate the effects of activation of the CRF2R on skeletal muscle-related physiological processes. Administration of urocortin II to mice prevented the loss of skeletal muscle mass resulting from disuse due to casting, corticosteroid treatment, and nerve damage. In addition, urocortin II treatment prevented the loss of skeletal muscle force and myocyte cross-sectional area that accompanied muscle mass losses resulting from disuse due to casting. Finally, we observed increased skeletal muscle mass and force in normal muscles when mice are treated with urocortin II. These results were confirmed using two additional CRF2R agonists, urocortin I and sauvagine. Thus, activation of the CRF2R modulates skeletal muscle mass in both normal and atrophying muscle. Therefore, CRF2R-selective agonists may find utility in the treatment of skeletal muscle wasting diseases including age-related muscle loss or sarcopenia.

. Activation of the CRF 2 receptor modulates skeletal muscle mass under physiological and pathological conditions. Am J Physiol Endocrinol Metab 285: E889-E898, 2003; 10.1152/ajpendo.00081.2003.-Two receptors activated by the corticotropin-releasing factor (CRF) family of peptides have been identified, the CRF 1 receptor (CRF1R) and the CRF 2 receptor (CRF2R). Of these, the CRF2R is expressed in skeletal muscle. To understand the role of the CRF2R in skeletal muscle, we utilized CRFR knockout mice and CRF2R-selective agonists to modulate nerve damage and corticosteroid-and disuseinduced skeletal muscle atrophy in mice. These analyses demonstrated that activation of the CRF2R decreased nerve damage and corticosteroid-and disuse-induced skeletal muscle mass and function loss. In addition, selective activation of the CRF2R increased nonatrophy skeletal muscle mass. Thus we describe for the first time a novel activity of the CRF2R, modulation of skeletal muscle mass. atrophy; hypertrophy; corticotropin-releasing factor receptor; sauvagine; urocortin II CORTICOTROPIN-RELEASING FACTOR (CRF), also known as corticotropin-releasing hormone (CRH), and its functionally and structurally related analogs are known to have many physiological functions, including the coordination of the endocrine, autonomic, behavioral, and immune responses to stress. These include, for example, increased secretion of epinephrine, norepinephrine, mineralocorticoids, corticosteroids, somatostatin, dynorphin, ␤-endorphin, ACTH, and glucose and decreased secretion of gonadotropin-releasing hormone, luteinizing hormone, follicle-stimulating hormone, and melatonin; increased arousal and emotional reactivity, including increased locomotion, sniffing, grooming, and rearing; decreased sexual behavior and reproductive potential; decreased feeding and gastrointestinal functionality; increased heart rate and mean arterial blood pressure by central administration or decreased heart rate and mean arterial blood pressure by peripheral administration; neuronal activity modulation; and altered immune system activity (11,13,19,43). CRF and related functional/structural analogs mediate their effects through CRFRs via both neuronal (as putative neurotransmitters) and neuroendocrine pathways.

A prominent feature of long-term regulation of the alpha2A-adrenergic receptor (alpha2AAR) is a loss of cellular receptors over time (downregulation). The molecular determinants of downregulation were sought by targeting regions of the receptor involved in G protein coupling and phosphorylation. Mutated receptors, consisting of chimeric substitutions of analogous beta2-adrenergic receptor (beta2AR) and serotonin 5-hydroxytryptamine1A (5-HT1A) receptor sequence into the second intracellular loop (ICL2) (residues 113-149), the amino terminus (residues 218-235) and carboxy terminus (residues 355-371) of ICL3, and a deletion of the beta-adrenergic receptor kinase (betaARK) phosphorylation sites in the third intracellular loop (ICL3) (residues 293-304), were expressed in Chinese hamster ovary (CHO) cells. Wild-type alpha2AAR underwent 31% +/- 3% downregulation after 24 h of exposure to 100 microM epinephrine. Loss of downregulation was observed with some mutants, but this was not related to functional coupling to inhibitory or stimulatory guanine nucleotide regulatory binding proteins (Gi or GS) or to phosphorylation. Rather, any mutant with a substitution of the amino terminus of ICL3 (regardless of whether the substitution was with beta2AR or 5-HT1A sequence) resulted in upregulation. Studies with an inhibitor of protein synthesis indicated that the primary mechanism of downregulation of the alpha2AAR is agonist-promoted degradation of receptor protein which requires a destabilization sequence in the amino terminus of ICL3. Thus, in contrast to other G protein-coupled receptors, in which G protein coupling or phosphorylation are critical for long-term agonist regulation, the alpha2AAR has a specific structural domain distinct from these other functional regions that serves to direct agonist-promoted downregulation.