Mutations in the growth hormone receptor (GHR) gene can cause growth hormone (GH) resistance. Given the sequence homology between the extracellular domain of the GHR and a soluble GH-binding protein (GH-BP), it is remarkable that GH-BP binding activity is absent from the serum of patients with Laron-type GH insensitivity, a hereditary form of severe dwarfism. We have previously identified a mutation within the extracellular domain of this receptor, replacing phenylalanine by serine at position 96 of the mature protein, in a patient with Laron syndrome. We have now investigated the effect of this Phe -* Ser substitution on hormone binding activity by expressing the total human GHR cDNA and mutant form in eukaryotic cells. The wild-type protein expressed was able to bind GH but no plasma membrane binding was detectable on cells transfected with the mutant cDNA; this was also the case of cells transfected with a Phe"-Ala mutant cDNA, suggesting that the lack of binding activity is not due to a posttranslational modification of serine. Examination of the variant proteins in subcellular fractions revealed the presence of specific GH binding activity in the lysosomal fraction, whereas immunofluorescence studies located mutant proteins in the cytosol. Our findings suggest that these mutant GHIRs fail to follow the correct intracellular transport pathway and underline the potential importance of this phenylalanine residue, which is conserved among the GH, prolactin, and erythropoietin receptors that belong to the same cytokine receptor superfamily.The cloning of a human growth hormone receptor (hGHR) cDNA (1) has provided a valuable tool for investigating GHR structure-function relationships at the molecular level. However, examination of the complete amino acid sequence deduced from this cDNA clone has failed to identify functionally important regions. One approach to this problem is to study diseases, from which clues on normal physiology can be gleaned. As a first step toward this goal, we showed that mutations in the GHR gene can be associated with Laron syndrome (2), an autosomal recessive GH-resistance disorder (3, 4). This finding was particularly interesting since the amino-terminal amino acid sequences of GHR and serum GH-binding protein (GH-BP) (5) in various species are identical, suggesting that GH-BP is derived from the extracellular binding region of the receptor (6, 7), whereas serum from patients with Laron syndrome lacks functional GH-BP (8). Overall, these data suggested that GH resistance in this disease could result from defective hGHR binding.While investigating one such patient with GH resistance, we identified a mutation within the extracellular domain, replacing a phenylalanine by a serine at position 96 (2).Although this Phe' is conserved through evolution in all members of this class of transmembrane receptors (including GH, prolactin, and erythropoietin receptors in all species studied so far) (9), its functional importance in regulating the binding activity of GHR remained to be demon...