1. IGF in circulation The majority (> 75 %) exists as bound form –IGF binding proteins (IGFBPs) IGFBPs –6 proteins and several related proteins –Serum IGFBP IGFBP3 Largest IGFBP (150 kDa complex) Regulated by GH –Other IGFBPs Smaller IGFBPs Regulation of IGF cellular uptake

3. IGFBP3 –Three components IGF IGFBP3 Acid-labile subunit

4. Function of IGFBPs –Prolongs half-life of IGFs Minutes to hours IGFBP3 –Regulation of IGF bioavailability Amount of IGFs being uptaken by the cell –Proteolytic cleavage

5. Growth hormone receptor

6. Receptors with associated kinase –Type I cytokine receptor GH and PRL No intrinsic kinase activity Intracellular domain –Interacts with tyrosine kinases Janus tyrosine kinase (JAK)

7. Activation of receptor –Dimerization One hormone, two receptors –Interaction of receptors with GH Two different site (1 and 2) with different affinity –Site 1 has a higher affinity to GHR

8. Activation of receptor –Activation of JAK via phosphorylation Activated JAK –Phosphorylate intracelluar domain of the receptor (tyrosine residues) Serve as a docking site for other intracellular proteins –Signal transducer and activator of transcription (STATS) –Activated by phosphorylation Activated STATS –Dimerization –Enter nucleus and interact with DNA

10. Distribution of GHR GHR –Expressed in all tissues Degree of expression varies greatly among tissues –Greatest expression Liver –Regulation of GHR Regulated at mRNA level

11. Bovine GHR Gene Structure 1B1C1A2345768910 1A2345768910 23457689 1C 23457689101B GHR 1A GHR 1B GHR 1C 87 Kb

12. Differential expression of GHR mRNA –Different promoters that regulate expression of GHR mRNA –Two types Liver-specific Constitutive

13. Liver-specific GHR promoter –Prototypical promoter TATA box Many binding sites for nuclear factors Very specific transcription start site

14. Constitutive GHR promoters –No TATA box –Some sites for nuclear proteins to interact No specific regulation –Different transcription start sites

15. Several different promoters –Several different mRNAs Identical protein Difference –5’ UTR (exon 1) –Exons 2-10 (ORF) are identical among different mRNA transcripts Translational efficiency –Greater in liver-specific transcript

16. Inhibition of GHR signaling –Suppressor of cytokine signaling (SOCS) Inhibitors of JAK/STAT signaling –Inhibition of kinase activity –Inhibition of phosphorylation of JAK and STAT –Reduced STAT dimerization and translocation

17. IGF receptors Two types –Type I Insulin receptor superfamily Binds equally well to IGF-I and IGF-II –Type II Mannose-6-phosphate receptor Only binds to IGF-II

20. Type I IGF receptor Two components –Alpha subunit Extracellular domain –Beta subunit Intracellular Receptor tyrosine kinase –Beta subunit Intrinsic kinase activity

21. Two subuints –Generated as one large protein Proteolytic cleavage Linked together by one disulfide bridge Combination of two sets of two subunits –Disulfide bridge between two alpha subunits

23. IGF-II receptor Mannose-6-phosphate receptor –Specifically interacts with IGF-II but not IGF-I –No intracellular signaling domain No signal transduction –Increase cellular uptake of IGF-II Regulation of bioactivity and bioavailability