Insulin-like growth factor-I (IGF-I) is a major hormone that is required not only for proper growth during child development, but remarkably is also negatively associated with longevity. Circulating levels of IGF-I increase considerably during childhood, then decline after the 6th decade of life and are particularly low in centenarians. Primary IGF deficiency (PIGFD) early in life severely retards growth and affects metabolism, while at the other end of human life, low IGF-I seems beneficial as it is associated with low incidence of diabetes and cancer, among other effects on longevity. Production of IGF-I is controlled at the transcriptional level by complex endocrine regulatory circuits; however, we found that in addition biosynthesis of the hormone precursor relies on the stress protein GRP94. IGF-dependent processes, such as muscle differentiation or responses to metabolic stress are strictly dependent on the activity of GRP94 and are curtailed when GRP94 activity limits IGF production. Given this molecular link, we hypothesize that human GRP94 alleles have differential activity and thereby affect the variation in production of IGF-I. Hypomorphic alleles are expected to be over- represented in populations with very low IGF-I levels, and are proposed to become limiting when the demand for IGF-I is high. This proposal will test this hypothesis as follows: We will sequence the GRP94 gene from three cohorts with low IGF-I: one from children with PIGFD and two of old people and centenarians (Aim 1). To date, two coding mutations have been identified in the former and one in the latter population. The functionality of the coding variants identified will be determined using a novel cell-based assay, where the survival of GRP94-deficient cells after serum withdrawal is rescued when supplemented with functional GRP94 that allows for proper IGF processing and secretion (Aim 2). Use of this assay has shown that one mutation from the PIGFD cohort and one from the longevity cohort are hypomorphic when compared to wild type GRP94. The functionality of hypomorphic GRP94 variants will be further tested in a mouse model (Aim 3), in which expression of the endogenous protein in skeletal muscle will be replaced with a human GRP94 transgene. Deletion of the endogenous gene in this model results in decreased production of muscle IGF-I, leading to smaller animals with smaller muscles. We expect hypomorphic human GRP94 to be insufficient for full development of the musculature, and we will ask whether the other, whole body and metabolic effects are also evident when GRP94 activity is limiting in the muscle. This animal will model aspects of PIGFD as well as muscle atrophy, processes that are important for the quality of human aging. Relevance to Human Health: This study will extend the correlation of human genetic variability with phenotype and will assign causal function to the various alleles of GRP94 by using both cell culture and in vivo models. Success of this work will pave the way for developing new ways to modulate IGF production in both children with PIGFD as well as during aging.