The mutant growth hormone-releasing hormone (GHRH) receptor of the little mouse does not bind GHRH

The little mouse is a dwarf strain characterized by low levels of GH, pitui tary hypoplasia, and an unresponsiveness to treatment with exogenous GHRH. The defect has been mapped to a missense mutation in the GHRH receptor gene that abolishes the function of the receptor, but the mechanism of this ina ctivation is unknown. Receptor function might be affected at the level of p rotein expression, maturation and processing, localization to the cell surf ace, Ligand binding, or signaling. In this study, Western blots, using anti serum raised against the GHRH receptor and immunoprecipitation analysis of epitope-tagged receptors, demonstrate that both wild-type and mutant recept or proteins are expressed at equivalent levels in transfected cells. Immuno fluorescence analysis of intact and permeabilized cells expressing the epit ope-tagged receptors suggests that wild-type and little mouse receptors are similarly localized to the cell surface. A species homologous binding assa y was developed and used to show that I-125-mouse GHRH binds with high affi nity to the wild-type mouse receptor but not to the little mutant receptor. Consistent with this, the mutant receptor fails to stimulate intracellular cAMP accumulation. Our results demonstrate that the little mutation does n ot dramatically affect the expression level, glycosylation, or cellular loc alization of the receptor protein but that it blocks specific GHRH binding, and therefore, signaling does not take place.