Pheochromocytoma in NF1 occurs in older patients with no family history compared to other syndromes; it is mostly unilateral, secretory and benign. The older age at diagnosis of PHEO could be secondary to delay in identification due to lack of systematic screening for PHEO in NF1.

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Previous research on growth hormone-releasing factor analogues has used pituitary cell culture assay systems to evaluate in vitro their biological activity. However, binding assay systems in which receptor affinity and peptide stability can be assessed independently have been lacking so far. Since we have recently developed a sensitive GRF binding assay with [125I-Tyr10]hGRF(1-44)NH2, this method was applied to structure-affinity studies as a first step of screening GRF analogues. Acylation of the N-terminus of hGRF(1-29)NH2 generally decreased its affinity (relative affinity to hGRF(1-29)NH2 (RA), 26-85%). Replacement of the C-terminal carboxamide by a free carboxylic function decreased affinity likely by diminishing its proteolytic stability (RA, 57%). Removal of Tyr1, Ser9, Lys12, Val13, Gly15, Gln16, or Lys21 drastically decreased its affinity (RA, less than 3%). Multiple amino acid deletions in the segment 13-21 of hGRF(1-29)NH2 also led to a loss of affinity as did replacing segment 13-15, 16-18, or 19-21 by an octanoyl moiety (RA, less than 1%). Removal of Asn8, Gln24, Asp25, Ile26, Met27, and Ser28 or Arg29 had less effect on GRF receptor affinity (RA, 5-33%). Removal of Met27 or Ser28 only slightly affected hGRF(1-29)NH2 affinity (RA, 62-78%). Altogether, these results indicate that the amino acids contained in the segment 13-21 are more important than those of 24-29 to insure high affinity receptor binding or to maintain an optimal conformation to allow GRF binding.

A site-directed polyclonal antipeptide antibody was generated in rabbits against segment 392–404 of the rat pituitary growth hormone-releasing hormone receptor (GHRH-R), using a multiple antigenic peptide system strategy of immunization. This C-terminal intracellular region of the rat GHRH-R exhibits 85% sequence identity with the human GHRH-R. The purified anti-GHRH-R(392–404) IgGs were characterized in cell lines expressing the human GHRH-R and in rat and human anterior pituitary, using immunoblotting. The polyclonal antibody recognized a 45-kD protein in human GHRH-R-transfected BHK 570 cell membrane preparations but not in wild-type cells. A 45-kD N^α-tagged human GHRH-R was immunodetected with both antitag and anti-GHRH-R antibodies in human GHRH-R-transfected HEK 293 cells. Cross-linking of [¹²⁵I-Tyr¹⁰]hGHRH(1–44)NH₂ to GHRH-R-transfected BHK cells led to the detection of a major and specific 45-kD radioactive complex. Its probing with the anti-GHRH-R(392–404) IgGs led also to the detection of a 45-kD entity. In rat anterior pituitary homogenates or membrane preparations, immunoblotting led to the detection of 44-, 47- and 65-kD proteins. In human anterior pituitary membrane preparations, immunoblotting led to the detection of 52- and 55-kD proteins. No immunoreactive signal was observed in the rat liver. Cross-linking of [¹²⁵I-Tyr¹⁰]hGHRH(1–44)NH₂ to rat anterior pituitary homogenates revealed the presence of specific 28-, 47- and 65-kD radioactive complexes. Probing of these radioactive complexes with the anti-GHRH-R(392–404) IgGs resulted in the visualization of 28-, 47- and 65-kD entities and of an additional immunoreactive 44-kD protein. To assess the usefulness of this GHRH-R antibody, estimation of changes in the concentration of rat anterior pituitary GHRH-R was performed by immunoblotting and compared to binding data after a 3-week antithyroid treatment. The treatment known to depress the 2.5- and 4-kb GHRH-R mRNA transcripts by at least 1.7-fold decreased the apparent maximal concentration of high (B_max1) and low (B_max2) affinity binding sites by 4.6- and 15.2-fold, respectively, and the 47- and 65-kD GHRH-R proteins by 3.5- and 1.25-fold, respectively. Altogether, the characteristics of the anti-GHRH-R(392–404) polyclonal antibody indicate that it specifically recognizes the human and rat GHRH-R. It also represents an additional valuable tool to estimate variations of GHRH-Rs in physiopathological conditions known to affect GHRH-R mRNA and/or GHRH binding site concentrations.