GH receptor (GHR) undergoes regulated proteolysis by both metalloprotease (alpha-secretase) and gamma-secretase activities. alpha-Secretase activity regulates GHR availability and sensitivity and generates circulating GH binding protein. The function of gamma-secretase cleavage is yet uncertain. We investigated GHR determinants that affect inducible sequential alpha- and gamma-secretase cleavage and thus remnant and stub generation, respectively. Purification and N-terminal sequencing of the stub revealed that gamma-secretase cleavage occurs at an epsilon-site in GHR's transmembrane domain four residues from the intracellular domain. Mutagenesis revealed that deletion of the proximal two transmembrane residues prevented both alpha- and gamma-secretase-mediated proteolysis and deletion of four residues around the epsilon-site precluded surface GHR expression and proteolysis. However, point mutations in and around the epsilon-site affected neither alpha- or gamma-secretase cleavage. We conclude that both cleavages likely occur at the cell surface and sequentially (alpha-secretase followed by gamma-secretase) and that epsilon-site cleavage by gamma-secretase does not require a consensus sequence. (C) 2011 Elsevier Inc. All rights reserved.