Overexpression of the Short Form of the Growth Hormone Receptor in 3T3-L1 Mouse Preadipocytes

Abstract

In rodents, the gene for the growth hormone receptor (GHR) gives rise to two mRNA transcripts encoding two proteins: a larger membrane spanning receptor (GHR_l) and a smaller isoform, GHR_s that consists of the extracellular domain and a unique hydrophilic carboxyl terminus. We examined the hypothesis that GHR_s may contribute to cellular binding of GH and play a role in growth hormone (GH) signaling. Rat cDNA encoding GHR_s was ligated into the mammalian expression vector pcDNAl/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 GHR_s 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 GHR_l in extracts of these cells was unchanged, indicating that increased binding could not be accounted for by effects on formation or degradation of GHR_l. The transfected cDNA for GHR_s directs the synthesis of a 50 kDa protein. Cross-linking of [¹²⁵l]hGH to transfected 3T3-L1 cells indicated a 3.5-fold increase in a 72 kDa GHR_s[¹²⁵l]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 ~70% in control cells, but in the transfected cells, protein synthesis was reduced only by ~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 ~70% in control cells, but only by ~30% in cells with increased GH binding. One nanomolar IGF-1 restored 2DG uptake to levels seen after 2 hr of 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 GHR_s produce greater amounts of IGF-1 than control cells or transfected clones that failed to overexpress GHR_s. We conclude that GHR_s contributes to GH binding and may therefore be a functional receptor. In addition, overexpression of GHR_s in 3T3-L1 cells altered cell function in the absence of GH.