Growth hormone activity in mitochondria depends on GH receptor Box 1 and involves caveolar pathway targeting

Growth hormone (GH) binding to its receptor (GHR) initiates GH-dependent signal transduction and internalization pathways to generate the biological effects. The precise role and way of action of GH on mitochondrial function are not yet fully understood. We show here that GH can stimulate cellular oxygen consumption in CHO cells transfected with cDNA coding for the full-length GHR. By using different GHR cDNA constructs, we succeeded in determining the different parts of the GHR implicated in the mitochondrial response to GH. Polarography and two-photon excitation fluorescence microscopy analysis showed that the Box 1 of the GHR intracellular domain was required for an activation of the mitochondrial respiration in response to a GH exposure. However, confocal laser scanning microscopy demonstrated that cells lacking the GHR Box 1 could efficiently internalize the hormone. We demonstrated that internalization mediated either by clathrin-coated pits or by caveolae was able to regulate GH mitochondrial effect: these two pathways are both essential to obtain the GH stimulatory action on mitochondrial function. Moreover, electron microscopic and biochemical approaches allowed us to identify the caveolar pathway as essential for targeting GH and GHR to mitochondria.     

Activation of insulin receptor signaling by a single amino acid substitution in the transmembrane domain.

The insulin receptor is a ligand-activated tyrosine kinase composed of two alpha and two beta subunits. A single transmembrane domain composed of 23 hydrophobic residues is contained in each beta subunit. We examined the role of the transmembrane domain in regulating insulin receptor signaling by inserting a negatively charged amino acid (Asp) for Val938 (V938D). Chinese hamster ovary (CHO) cells were stably transfected with a plasmid containing both the neomycin-resistance gene and either the wild-type or the mutant (V938D) insulin receptor cDNA. Insulin binding increased similarly in CHO cells stably transfected with the wild-type and the V938D-mutant insulin receptor cDNA. Insulin stimulated glucose transport and cell growth in cells expressing the normal insulin receptor. By contrast, in the absence of insulin, glucose transport and cell growth in CHO-V938D cells were as high as in insulin-stimulated control cells and no longer responsive to insulin stimulation. Phosphorylation of the beta subunit of the insulin receptor was also increased in CHO-V938D cells not exposed to insulin. These results support an essential role of the transmembrane domain of the insulin receptor in the transduction of insulin signaling.     

Growth hormone (GH) induction of tyrosine phosphorylation and activation of mitogen-activated protein kinases in cells transfected with rat GH receptor cDNA.

The mechanism of growth hormone (GH) action was studied in Chinese hamster ovary (CHO) cells transfected with GH receptor cDNA. Cytosolic extracts from GH- or phorbol ester (12-O-tetradecanoyl 4 beta-phorbol 13-acetate)-treated cells, transfected with full-length GH receptor cDNA, had an enhanced ability to phosphorylate myelin basic protein. Myelin basic protein, a substrate for mitogen-activated protein (MAP) kinase, was maximally phosphorylated using extracts from cells treated with 50 nM bovine GH for 10 min. In addition, GH treatment resulted in an increased cell proliferation by 30-60%. GH and 12-O-tetradecanoyl 4 beta-phorbol 13-acetate cause tyrosine phosphorylation of two proteins with M(r) of 40,000 and 42,000 that are also recognized by MAP kinase antibodies. These proteins were identified as MAP kinases by analyzing phosphotyrosine immunoprecipitates on Western blots using MAP kinase antibodies. In addition, GH induces mitogenicity, as well as MAP kinase activation, in CHO cells expressing a receptor in which 184 amino acids had been deleted in the carboxyl-terminal part of the intracellular domain. No GH effects were seen in untransfected cells, in CHO cells expressing a truncated GH receptor containing only 5 of 349 amino acids in the intracellular domain, or in cells expressing the soluble GH-binding protein. In conclusion, our data show that GH treatment of CHO cells, reconstituted with GH receptors, initiates a phosphorylation cascade which includes MAP kinase.     

Functional promiscuity of squirrel monkey growth hormone receptor toward both primate and nonprimate growth hormones

Primate growth hormone (GH) has evolved rapidly, having undergone approximately 30% amino acid substitutions from the inferred ancestral eutherian sequence. Nevertheless, human growth hormone (hGH) is physiologically effective when administered to nonprimate mammals. In contrast, its functional counterpart, the human growth hormone receptor (hGHR), has evolved species specificity so that it responds only to Old World primate GHs. It has been proposed that this species specificity of the hGHR is largely caused by the Leu --> Arg change at position 43 after a prior His --> Asp change at position 171 of the GH. Sequence analyses supported this hypothesis and revealed that the transitional phase in the GH:GHR coevolution still persists in New World monkeys. For example, although the GH of the squirrel monkey has the His --> Asp substitution at position 171, residue 43 of its GHR is a Leu, the nonprimate residue. If the squirrel monkey truly represents an intermediate stage of GH:GHR coevolution, its GHR should respond to both hGH and nonprimate GH. Also, if the emergence of species specificity was a result of the selection for a more efficient GH:GHR interaction, then changing residue 43 of the squirrel monkey growth hormone receptor (smGHR) to Arg should increase its binding affinity toward higher primate GH. To test these hypotheses, we performed protein-binding assays between the smGHR and both human and rat GHs, using the surface plasmon resonance methodology. Furthermore, the effects of reciprocal mutations at position 43 of human and squirrel monkey GHRs are measured for their binding affinities toward human and squirrel monkey GHs. The results from the binding kinetic assays clearly demonstrate that the smGHR is in the intermediate state of the evolution of species specificity. Interestingly, the altered residue Arg at position 43 of the smGHR does not lead to an increased binding affinity. The implications of these results on the evolution of the GH:GHR interaction and on functional evolution are discussed.     

Identification of three adjacent amino acids of interleukin-2 receptor beta chain which control the affinity and the specificity of the interaction with interleukin-2.

The beta chain of the interleukin-2 (IL-2) receptor (IL-2R beta) and the interleukin-3 (IL-3) binding protein AIC2A are members of the family of cytokine receptors, which also includes the receptors for growth hormone (GHR) and prolactin. A four amino acid sequence of AIC2A has recently been shown to be critical for IL-3 binding. We analyze here the function of the analogous sequence of human IL-2R beta and identify three amino acids, Ser132, His133 and Tyr134, which play a critical role in IL-2 binding. We show that some mutant IL-2 proteins with substitutions of a critical Asp residue in the N-terminal alpha-helix bind the mutant IL-2R beta receptor with a higher affinity than the wild-type receptor. This suggests that the critical Asp34 in the ligand and the sequence Ser-His-Tyr (positions 132-134) in the receptor interact directly. On the double barrel beta-stranded structural model of cytokine receptors, the residues important for ligand binding in IL-2R beta, AIC2A and GHR map to strikingly similar locations within a barrel, with the interesting difference that it is the N-terminal barrel for GHR and the C-terminal barrel for IL-2R beta and AIC2A.     

中华鲟促性腺激素释放激素受体基因cDNA序列的克隆及表达分析

为了研究中华鲟(Acipenser sinensis)促性腺激素释放激素受体(Gonadotropin-releasing hormone receptor, GnRH-R)基因在中华鲟中的组织表达特征, 为中华鲟生长发育调控研究提供基础数据, 通过构建中华鲟(Acipenser sinensis)垂体的SMART cDNA质粒文库, 采用cDNA末端快速扩增(RACE), 克隆得到了中华鲟GnRH-R基因的cDNA全长序列。该序列全长1530 bp, 有478 bp的5′非翻译区, 579 bp的开放阅读框和473 bp的3′非翻译区, 共编码192个氨基酸, 其成熟多肽含有5个Ｎ连糖基化位点。通过和已知其他鱼类的GnRH-R基因进行氨基酸序列多重比对, 发现其与真鲷(Pagrus major)的同源性最高, 为76%, 与米氏叶吻银鲛(Callorhinchus milii)的同源性最低, 为39%。采用实时荧光定量PCR (Real time PCR)方法, 检测了GnRH-R的mRNA在中华鲟心、肝、脾、肾、肠道、精巢、肌肉及脑组织中的表达状况, 发现其在精巢中大量转录, 而在其他组织中则表达微弱。以上结果表明中华鲟GnRH-R基因在性腺发育特别是精子发生过程中可能起重要作用。     

Identification,characterization, and expressions profile analysis of growth hormone receptors (GHR1 and GHR2) in Hybrid grouper (Epinephelus fuscoguttatus ♀ × Epinephelus polyphekadion ♂)

Growth hormone is an essential hormone that plays essential roles in growth, metabolism, cellular differentiation, immunity and reproduction in fish, by means of the growth hormone receptors. The encoding cDNA growth hormone receptors (GHR1 and GHR2) were cloned and characterized from Hybrid grouper (Epinephelus fuscoguttatus♀ × Epinephelus polyphekadion♂). Sequence analysis of the cloned GHR1 was observed as containing 2176, which comprised an ORF of 1842 bp, 5 UTR of 6 bp and 3 UTR of 328 bp, with 612 amino acids encoding proteins, while GHR2 was observed as containing 1824 bp that encompassed an ORF of 708 bp, 5 UTR of 48 bp and 3 UTR of 1068 bp with 235 amino acids encoding proteins. Relative mRNA expression of GHR1 and GHR2 in the liver and muscle was found to be highest respectively. Our findings provide vital statistics of GHRs likely to play a significant role in the growth of the fish.     

豚鼠生长激素受体cDNA的克隆

报道了豚鼠肝生长激素受体（ＧＨＲ）的ｃＲＮＡ克隆和编码区序列。它由１８９９ｂｐ组成,编码６１０个氨基酸。此外,还报道豚鼠ＧＨＲ的结构特征和同源性比较的结果。     

Identification and Characterization of GH Receptor and Serum GH-binding Protein in Chinese Sturgeon （Acipenser sinensis）

Growth hormone (GH) can stimulate bone and carti-lage cell proliferation and influence carbohydrate and lipidmetabolism. The binding of GH to its specific receptor(GHR) on the surface of target cells will induce dimeriza-tion of GHR, which allows the cytoplasmic region of GHRto interact and trigger downstream signaling and geneexpression [1,2]. GHR belongs to the cytokine receptor superfamily, andis expressed in many tissues such as the liver, muscle,adipose tissue, cartilage, and brain…     

Growth hormone receptor ubiquitination coincides with recruitment to clathrin-coated membrane domains

Endocytosis of the growth hormone receptor (GHR) depends on a functional ubiquitin conjugation system. A 10-amino acid residue motif within the GHR cytosolic tail (the ubiquitin-dependent endocytosis motif) is involved in both GHR ubiquitination and endocytosis. As shown previously, ubiquitination of the receptor itself is not required. In this paper ubiquitination of the GHR was used as a tool to address the question of at which stage the ubiquitin conjugation system acts in the process of GHR endocytosis. If potassium depletion was used to interfere with early stages of coated pit formation, both GHR endocytosis and ubiquitination were inhibited. Treatment of cells with methyl-beta-cyclodextrin inhibited endocytosis at the stage of coated vesicle formation. Growth hormone addition to methyl-beta-cyclodextrin-treated cells resulted in an accumulation of ubiquitinated GHR at the cell surface. Using immunoelectron microscopy, the GHR was localized in flattened clathrin-coated membranes. In addition, when clathrin-mediated endocytosis was inhibited in HeLa cells expressing a temperature-sensitive dynamin mutant, ubiquitinated GHR accumulated at the cell surface. Together, these data show that the GHR is ubiquitinated at the plasma membrane, before endocytosis occurs, and indicate that the resident time of the GHR at the cell surface is regulated by the ubiquitin conjugation system together with the endocytic machinery.     

Isolation, characterization, and distribution of two cDNAs encoding for growth hormone receptor in rainbow trout (Oncorhynchus mykiss)

Growth hormone (GH) plays important roles in a vast array of physiological processes, including growth, metabolism, and reproduction. In this study, cDNAs for two unique growth hormone receptor variants were cloned and sequenced from rainbow trout. The two cDNAs, one consisting of 2920 bp and the other of 2820 bp, share 87.2% identity in nucleotide sequence and 85.5% identity in deduced amino acid sequence and presumably arose through gene duplication. The cDNAs encode for putative 593- and 594-amino acid growth hormone receptors (designated GHR1 and GHR2, respectively), each containing a single transmembrane domain and other motifs characteristic of the receptor family. Both GHR1 and GHR2 mRNAs were present in all tissues examined. Trout GHR mRNAs are differentially expressed, both in terms of abundance among tissues and in terms of abundance within selected tissues. GHR1 was more abundant than GHR2 in the brain, whereas GHR2 was more abundant than GHR1 in pancreas and spleen. These findings expand our understanding of the evolution of the GH receptor family and suggest that independent mechanisms serve to regulate the tissue-specific expression of GHR mRNAs.     

尼罗罗非鱼生长激素及其受体的cDNA克隆与mRNA表达的雌雄差异

尼罗罗非鱼(Oreochromis niloticus)雌雄鱼生长差异明显,为了探讨其原因,本文采用RT-PCR方法克隆了尼罗罗非鱼生长激素(Growthhormone,GH)及其受体(Growth hormone receptor,GHR)的cDNA序列,并应用半定量RT-PCR方法比较了雌、雄尼罗罗非鱼垂体GHmRNA、肝脏GHRmRNA、肌肉GHRmRNA的表达差异。序列分析表明:GH开放阅读框为615bp,共编码204个氨基酸;GHR开放阅读框为1908bp,共编码635个氨基酸。以RT-PCR方法研究了GH、GHR在各组织的分布情况,结果表明:GH仅在垂体中检测到有表达,而GHR在所检测的18种组织中均有表达,其中以肝脏、肌肉、性腺、下丘脑、胸腺表达量较高。以半定量RT-PCR方法进一步比较了雌、雄尼罗罗非鱼垂体GHmRNA、肝脏GHRmRNA、肌肉GHRmRNA的表达量,结果表明:雄鱼垂体GHmRNA和肝脏GHRmRNA的表达量均显著高于雌鱼,肌肉GHRmRNA的表达量则无显著差异,推测垂体GHmRNA和肝脏GHRmRNA表达的雌雄差异是尼罗罗非鱼雌雄生长差异的主要原因之一。     

Molecular cloning and pharmacological characterization of the canine B1 and B2 bradykinin receptors

The dog is a valuable animal model in the study of the physiological role of both the B1 and B2 bradykinin receptors. To more thoroughly characterize the pharmacological properties of the canine kinin receptors we isolated the cDNA sequence encoding the B1 and B2 bradykinin receptor subtypes and overexpressed them in Chinese hamster ovary (CHO) cells. The cDNA sequence of the canine B1 bradykinin receptor encodes a protein comprised of 350 amino acids that is 76% identical to the human B1 bradykinin receptor. The cDNA sequence of the canine B2 bradykinin receptor encodes a protein of 392 amino acids that is 81% identical to the human B2 bradykinin receptor. The amino acid sequence of the canine B1 and B2 receptors are 35% identical. Pharmacological studies of the cloned receptors revealed that the agonist affinity of the dog B1 receptor is similar to the rodent B1 receptors, and differs from the human form in that there is no preference for the presence of the N-terminal Lys residue of [des-Arg10]Lys-bradykinin. Significantly, the B1 receptor antagonist [des-Arg9,Leu8]BK behaves as partial agonist on the cloned dog B1 receptor. The dog B2 receptor exhibits the 'classical' pharmacological properties of this receptor subtype.     

Chromosome mapping of the growth hormone receptor gene in man and mouse

Pituitary growth hormone (GH) is essential for normal growth and development in animals and GH deficiency leads to dwarfism. This hormone acts via specific high-affinity cell surface receptors found in liver and other tissues. The recent cloning and sequencing of cDNAs encoding human and rabbit GH receptors (GHR) has demonstrated that this receptor is unrelated to any previously described cell membrane receptor or growth factor receptor. We have used the cloned human GHR cDNA to map the GHR locus to the proximal short arm of human chromosome 5, region p13.1----p12, and to mouse chromosome 15 by Southern blot analysis and in situ hybridization. While human chromosome 5 carries several genes for hormone and growth factor receptors, GHR is the only growth-related gene so far mapped to the short arm. Inasmuch as GHR is the first gene with apparently homologous loci on human chromosome 5 and mouse chromosome 15, it identifies a new homologous conserved region. In humans, deficiency of GH receptor activity probably causes Laron-type dwarfism, an autosomal recessive disorder prevalent in Oriental Jews. In mice, the autosomal recessive mutation miniature (mn) is characterized by severe growth failure and early death and has been mapped to chromosome 15. Our assignment of Ghr to mouse chromosome 15 suggests this as a candidate gene for the mn mutation.     

The ubiquitin conjugation system is required for ligand-induced endocytosis and degradation of the growth hormone receptor.

The ubiquitin-dependent protein degradation system has recently been implicated in downregulation of signal transducing receptors. Growth hormone receptor (GHR) cDNA was transfected into Chinese hamster ovary cells, which exhibit a temperature-sensitive defect in ubiquitin conjugation (CHO-ts20), as well as into wild-type cells (CHO-E36). Upon binding of growth hormone (GH), two GHR polypeptides dimerize and initiate signal transduction. In CHO-E36 and in CHO-ts20 at the permissive temperature the GHR was ubiquitinated and degraded in a GH-dependent fashion. However, at the non-permissive temperature in CHO-ts20 cells, neither GH-dependent uptake nor degradation of the GHR was observed, while in CHO-E36 cells both GHR uptake and degradation were accelerated. Incubation of CHO-E36 cells with inhibitors of endosomal/lysosomal function (NH4Cl, bafilomycin A1) markedly reduced ligand-induced GHR degradation. Our results indicate that a functional ubiquitin conjugating system is required for GH-induced endocytosis and that degradation of both the exoplasmic and cytoplasmic portions of the GHR occurs within the endosomal/lysosomal compartment.     

生长激素受体的研究进展

生长激素（GH）在促进动物生长、发育等代谢过程中起着重要作用,GH发挥生理作用的第一步是与靶细胞膜表面的生长激素受体（CHR）结合。现已基本阐明了CHR的结构及由CHR介导的信号转导途径,对GHR基因表达调节的机制也有了一定的了解。GHR是由约620个氨基酸组成的单链跨膜糖蛋白,其胞外区、跨膜区及胞区内分别由约245、25及350个氨基酸组成。由GHR介导的信号转导途径主要有：①酪氨酸激酶系统;②蛋白激酶C途径;③胰岛素受体底物途径。营养状况及GH等内分泌因子对GHR的表达也有调节作用。     

Jak2 and proteasome activities control the availability of cell surface growth hormone receptors during ligand exposure

Several mechanisms participate in the down-regulation of growth hormone receptor (GHR) signalling under ligand exposure. In CHO cells expressing GHR, we show that ligand stimulation induces degradation of the total cell GHR content. Experiments with 125I-hGH indicate that ligand-bound internalized receptors are not immediately replaced. Using cell surface biotinylation, we demonstrate for the first time that, concomitantly with the degradation of cell surface receptors, GHRs from the intracellular compartments are also degraded. We thus suggest that under prolonged ligand exposure, some GHRs are targeted to the cell surface, while others are routed to degradation compartments. Inhibitors of Jak2 and of the proteasome partially inhibited degradation of cell surface receptors, while these compounds completely inhibit the degradation of intracellular GHRs, resulting in their accumulation. We therefore propose that Jak2 and proteasome activities control the amount of intracellular GHRs, and thus the availability of receptors at the cell surface, during ligand exposure.