Bick T, Frick G P, Leonard D, Leonard J L, Goodman H M
Department of Physiology, University of Massachusetts Medical School, Worchester 01655.
Proc Soc Exp Biol Med. 1994 Jul;206(3):185-9. doi: 10.3181/00379727-206-43739.
In rodents, the gene for the growth hormone receptor (GHR) gives rise to two mRNA transcripts encoding two proteins: a larger membrane spanning receptor (GHRL) and a smaller isoform, GHRs that consists of the extracellular domain and a unique hydrophilic carboxyl terminus. We examined the hypothesis that GHRs may contribute to cellular binding of GH and play a role in growth hormone (GH) signaling. Rat cDNA encoding GHRs was ligated into the mammalian expression vector pcDNA-I/neo and stably transfected into mouse 3T3-L1 preadipocytes which have endogenous GH receptors and, when differentiated into adipocytes, have the biochemical machinery to express the various GH effects. Sixteen of 24 neomycin resistant clones secreted at least twice as much GHRs in the growth medium as cells transfected with the vector alone, and in nine of these, GH binding was increased 2- to 4-fold. The amount of GHRL in extracts of these cells was unchanged, indicating that increased binding could not be accounted for by effects on formation or degradation of GHRL. The transfected cDNA for GHRs directs the synthesis of a 50 kDa protein. Cross-linking of [125I]hGH to transfected 3T3-L1 cells indicated a 3.5-fold increase in a 72 kDa GHRs[125I]hGH complex. In the presence of 10% newborn calf serum, incorporation of [35S]methionine into cellular proteins was similar in transfected clones and control cells. Deprivation of serum for 2 hr decreased protein synthesis by approximately 70% in control cells, but in the transfected cells, protein synthesis was reduced only by approximately 50% or 30% in cells exhibiting 2x or 3x increases in GH binding. In all cells, 1 nM IGF-1 restored protein synthesis to the serum replete level. Similarly, although 3H-2-deoxy-D-glucose (2DG) uptake was comparable in all cells after 2 hr of serum deprivation, 18 hr of serum deprivation decreased uptake by approximately 70% in control cells, but only by approximately 30% in cells with increased GH binding. One nanomolar IGF-1 restored 2DG uptake to levels seen after 2 hr or serum deprivation. IGF-1 had no effect after only 2 hr of serum deprivation. Measurement of IGF-1 secreted into the medium, revealed that clones which overexpress GHRs produce greater amounts of IGF-1 than control cells or transfected clones that failed to overexpress GHRs. We conclude that GHRs contributes to GH binding and may therefore be a functional receptor. In addition, overexpression of GHRs in 3T3-L1 cells altered cell function in the absence of GH.
在啮齿动物中，生长激素受体（GHR）基因产生两种mRNA转录本，编码两种蛋白质：一种较大的跨膜受体（GHRL）和一种较小的异构体GHRs，后者由细胞外结构域和独特的亲水性羧基末端组成。我们检验了这样一种假说，即GHRs可能有助于生长激素（GH）的细胞结合，并在生长激素信号传导中发挥作用。将编码GHRs的大鼠cDNA连接到哺乳动物表达载体pcDNA-I/neo中，并稳定转染到小鼠3T3-L1前脂肪细胞中，这些细胞具有内源性GH受体，并且在分化为脂肪细胞时，具有表达各种GH效应的生化机制。24个新霉素抗性克隆中有16个在生长培养基中分泌的GHRs至少是仅用载体转染的细胞的两倍，其中9个克隆的GH结合增加了2至4倍。这些细胞提取物中GHRL的量没有变化，这表明结合增加不能归因于对GHRL形成或降解的影响。转染的GHRs cDNA指导合成一种50 kDa的蛋白质。[125I]hGH与转染的3T3-L1细胞的交联表明，72 kDa的GHRs[125I]hGH复合物增加了3.5倍。在含有10%新生牛血清的情况下，转染克隆和对照细胞中[35S]甲硫氨酸掺入细胞蛋白的情况相似。血清剥夺2小时使对照细胞中的蛋白质合成减少约70%，但在转染细胞中，在GH结合增加2倍或3倍的细胞中，蛋白质合成仅减少约50%或30%。在所有细胞中，1 nM胰岛素样生长因子-1（IGF-1）将蛋白质合成恢复到血清充足水平。同样，尽管血清剥夺2小时后所有细胞中3H-2-脱氧-D-葡萄糖（2DG）摄取相当，但血清剥夺18小时使对照细胞中的摄取减少约70%，但在GH结合增加的细胞中仅减少约30%。1 nM IGF-1将2DG摄取恢复到血清剥夺2小时后所见的水平。血清剥夺仅2小时后，IGF-1没有作用。对分泌到培养基中的IGF-1的测量显示，过表达GHRs的克隆比对照细胞或未过表达GHRs的转染克隆产生更多的IGF-1。我们得出结论，GHRs有助于GH结合，因此可能是一种功能性受体。此外，3T3-L1细胞中GHRs的过表达在没有GH的情况下改变了细胞功能。
